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Alexander Varshavsky - One of the best experts on this subject based on the ideXlab platform.
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the arg n Degron pathway targets transcription factors and regulates specific genes
Proceedings of the National Academy of Sciences of the United States of America, 2020Co-Authors: Dylan C. Mitchell, Steven P. Gygi, Alexander VarshavskyAbstract: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.
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The Arg/N-Degron pathway targets transcription factors and regulates specific genes.
Proceedings of the National Academy of Sciences of the United States of America, 2020Co-Authors: Dylan C. Mitchell, Steven P. Gygi, Alexander VarshavskyAbstract: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.
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The ATF3 Transcription Factor Is a Short-Lived Substrate of the Arg/N-Degron Pathway
Biochemistry, 2020Co-Authors: Alexander VarshavskyAbstract:The Arg/N-Degron pathway targets proteins for degradation by recognizing their specific N-terminal residues or, alternatively, their non-N-terminal Degrons. In mammals, this pathway is mediated by ...
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the atf3 transcription factor is a short lived substrate of the arg n Degron pathway
Biochemistry, 2020Co-Authors: Alexander VarshavskyAbstract:The Arg/N-Degron pathway targets proteins for degradation by recognizing their specific N-terminal residues or, alternatively, their non-N-terminal Degrons. In mammals, this pathway is mediated by ...
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Recognition of nonproline N-terminal residues by the Pro/N-Degron pathway
Proceedings of the National Academy of Sciences of the United States of America, 2020Co-Authors: Cheng Dong, Alexander Varshavsky, Artem Melnykov, Shun-jia Chen, Sara Weirich, Kelly Sun, Albert Jeltsch, Jinrong MinAbstract:Eukaryotic N-Degron pathways are proteolytic systems whose unifying feature is their ability to recognize proteins containing N-terminal (Nt) degradation signals called N-Degrons, and to target these proteins for degradation by the 26S proteasome or autophagy. GID4, a subunit of the GID ubiquitin ligase, is the main recognition component of the proline (Pro)/N-Degron pathway. GID4 targets proteins through their Nt-Pro residue or a Pro at position 2, in the presence of specific downstream sequence motifs. Here we show that human GID4 can also recognize hydrophobic Nt-residues other than Pro. One example is the sequence Nt-IGLW, bearing Nt-Ile. Nt-IGLW binds to wild-type human GID4 with a K d of 16 μM, whereas the otherwise identical Nt-Pro-bearing sequence PGLW binds to GID4 more tightly, with a K d of 1.9 μM. Despite this difference in affinities of GID4 for Nt-IGLW vs. Nt-PGLW, we found that the GID4-mediated Pro/N-Degron pathway of the yeast Saccharomyces cerevisiae can target an Nt-IGLW-bearing protein for rapid degradation. We solved crystal structures of human GID4 bound to a peptide bearing Nt-Ile or Nt-Val. We also altered specific residues of human GID4 and measured the affinities of resulting mutant GID4s for Nt-IGLW and Nt-PGLW, thereby determining relative contributions of specific GID4 residues to the GID4-mediated recognition of Nt-Pro vs. Nt-residues other than Pro. These and related results advance the understanding of targeting by the Pro/N-Degron pathway and greatly expand the substrate recognition range of the GID ubiquitin ligase in both human and yeast cells.
Jaekwon Lee - One of the best experts on this subject based on the ideXlab platform.
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cadmium and secondary structure dependent function of a Degron in the pca1p cadmium exporter
Journal of Biological Chemistry, 2016Co-Authors: Nathan Smith, Wenzhong Wei, Miaoyun Zhao, Xiaojuan Qin, Javier Seravalli, Heejeong Kim, Jaekwon LeeAbstract:Protein turnover is a critical cellular process regulating biochemical pathways and destroying terminally misfolded or damaged proteins. Pca1p, a cadmium exporter in the yeast Saccharomyces cerevisiae, is rapidly degraded by the endoplasmic reticulum-associated degradation (ERAD) system via a cis-acting Degron that exists at the 250-350 amino acid region of Pca1p and is transferable to other proteins to serve as a degradation signal. Cadmium stabilizes Pca1p in a manner dependent on the Degron. This suggested that cadmium-mediated masking of the Degron impedes its interaction with the molecular factors involved in the ERAD. The characteristics and mechanisms of action of the Degron in Pca1p and most of those in other proteins however remain to be determined. The results presented here indicate that specific cysteine residues in a Degron of Pca1p sense cadmium. An unbiased approach selecting non-functional Degrons indicated a critical role of hydrophobic amino acids in the Degron for its function. A secondary structure modeling predicted the formation of an amphipathic helix. Site-directed mutagenesis confirmed the functional significance of the hydrophobic patch. Last, hydrophobic amino acids in the Degron- and cadmium-binding region affected the interaction of Pca1p with the Ssa1p molecular chaperone, which is involved in ERAD. These results reveal the mechanism of action of the Degron, which might be useful for the identification and characterization of other Degrons.
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Cadmium and Secondary Structure-dependent Function of a Degron in the Pca1p Cadmium Exporter *
The Journal of biological chemistry, 2016Co-Authors: Nathan Smith, Wenzhong Wei, Miaoyun Zhao, Xiaojuan Qin, Javier Seravalli, Heejeong Kim, Jaekwon LeeAbstract:Protein turnover is a critical cellular process regulating biochemical pathways and destroying terminally misfolded or damaged proteins. Pca1p, a cadmium exporter in the yeast Saccharomyces cerevisiae, is rapidly degraded by the endoplasmic reticulum-associated degradation (ERAD) system via a cis-acting Degron that exists at the 250-350 amino acid region of Pca1p and is transferable to other proteins to serve as a degradation signal. Cadmium stabilizes Pca1p in a manner dependent on the Degron. This suggested that cadmium-mediated masking of the Degron impedes its interaction with the molecular factors involved in the ERAD. The characteristics and mechanisms of action of the Degron in Pca1p and most of those in other proteins however remain to be determined. The results presented here indicate that specific cysteine residues in a Degron of Pca1p sense cadmium. An unbiased approach selecting non-functional Degrons indicated a critical role of hydrophobic amino acids in the Degron for its function. A secondary structure modeling predicted the formation of an amphipathic helix. Site-directed mutagenesis confirmed the functional significance of the hydrophobic patch. Last, hydrophobic amino acids in the Degron- and cadmium-binding region affected the interaction of Pca1p with the Ssa1p molecular chaperone, which is involved in ERAD. These results reveal the mechanism of action of the Degron, which might be useful for the identification and characterization of other Degrons.
Jinrong Min - One of the best experts on this subject based on the ideXlab platform.
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Molecular basis for ubiquitin ligase CRL2FEM1C-mediated recognition of C-Degron
Nature chemical biology, 2021Co-Authors: Xiaojie Yan, Xiaolu Wang, Mengqi Zhou, Lili Song, Jinrong Min, Cheng DongAbstract:Proteome integrity depends on the ubiquitin-proteasome system to degrade unwanted or abnormal proteins. In addition to the N-Degrons, C-terminal residues of proteins can also serve as degradation signals (C-Degrons) that are recognized by specific cullin-RING ubiquitin ligases (CRLs) for proteasomal degradation. FEM1C is a CRL2 substrate receptor that targets the C-terminal arginine Degron (Arg/C-Degron), but the molecular mechanism of substrate recognition remains largely elusive. Here, we present crystal structures of FEM1C in complex with Arg/C-Degron and show that FEM1C utilizes a semi-open binding pocket to capture the C-terminal arginine and that the extreme C-terminal arginine is the major structural determinant in recognition by FEM1C. Together with biochemical and mutagenesis studies, we provide a framework for understanding molecular recognition of the Arg/C-Degron by the FEM family of proteins.
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Recognition of nonproline N-terminal residues by the Pro/N-Degron pathway
Proceedings of the National Academy of Sciences of the United States of America, 2020Co-Authors: Cheng Dong, Alexander Varshavsky, Artem Melnykov, Shun-jia Chen, Sara Weirich, Kelly Sun, Albert Jeltsch, Jinrong MinAbstract:Eukaryotic N-Degron pathways are proteolytic systems whose unifying feature is their ability to recognize proteins containing N-terminal (Nt) degradation signals called N-Degrons, and to target these proteins for degradation by the 26S proteasome or autophagy. GID4, a subunit of the GID ubiquitin ligase, is the main recognition component of the proline (Pro)/N-Degron pathway. GID4 targets proteins through their Nt-Pro residue or a Pro at position 2, in the presence of specific downstream sequence motifs. Here we show that human GID4 can also recognize hydrophobic Nt-residues other than Pro. One example is the sequence Nt-IGLW, bearing Nt-Ile. Nt-IGLW binds to wild-type human GID4 with a K d of 16 μM, whereas the otherwise identical Nt-Pro-bearing sequence PGLW binds to GID4 more tightly, with a K d of 1.9 μM. Despite this difference in affinities of GID4 for Nt-IGLW vs. Nt-PGLW, we found that the GID4-mediated Pro/N-Degron pathway of the yeast Saccharomyces cerevisiae can target an Nt-IGLW-bearing protein for rapid degradation. We solved crystal structures of human GID4 bound to a peptide bearing Nt-Ile or Nt-Val. We also altered specific residues of human GID4 and measured the affinities of resulting mutant GID4s for Nt-IGLW and Nt-PGLW, thereby determining relative contributions of specific GID4 residues to the GID4-mediated recognition of Nt-Pro vs. Nt-residues other than Pro. These and related results advance the understanding of targeting by the Pro/N-Degron pathway and greatly expand the substrate recognition range of the GID ubiquitin ligase in both human and yeast cells.
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recognition of nonproline n terminal residues by the pro n Degron pathway
Proceedings of the National Academy of Sciences of the United States of America, 2020Co-Authors: Cheng Dong, Alexander Varshavsky, Jinrong Min, Artem Melnykov, Shun-jia Chen, Sara Weirich, Kelly Sun, Albert JeltschAbstract:Eukaryotic N-Degron pathways are proteolytic systems whose unifying feature is their ability to recognize proteins containing N-terminal (Nt) degradation signals called N-Degrons, and to target these proteins for degradation by the 26S proteasome or autophagy. GID4, a subunit of the GID ubiquitin ligase, is the main recognition component of the proline (Pro)/N-Degron pathway. GID4 targets proteins through their Nt-Pro residue or a Pro at position 2, in the presence of specific downstream sequence motifs. Here we show that human GID4 can also recognize hydrophobic Nt-residues other than Pro. One example is the sequence Nt-IGLW, bearing Nt-Ile. Nt-IGLW binds to wild-type human GID4 with a K d of 16 μM, whereas the otherwise identical Nt-Pro-bearing sequence PGLW binds to GID4 more tightly, with a K d of 1.9 μM. Despite this difference in affinities of GID4 for Nt-IGLW vs. Nt-PGLW, we found that the GID4-mediated Pro/N-Degron pathway of the yeast Saccharomyces cerevisiae can target an Nt-IGLW-bearing protein for rapid degradation. We solved crystal structures of human GID4 bound to a peptide bearing Nt-Ile or Nt-Val. We also altered specific residues of human GID4 and measured the affinities of resulting mutant GID4s for Nt-IGLW and Nt-PGLW, thereby determining relative contributions of specific GID4 residues to the GID4-mediated recognition of Nt-Pro vs. Nt-residues other than Pro. These and related results advance the understanding of targeting by the Pro/N-Degron pathway and greatly expand the substrate recognition range of the GID ubiquitin ligase in both human and yeast cells.
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Molecular basis of GID4-mediated recognition of Degrons for the Pro/N-end rule pathway.
Nature chemical biology, 2018Co-Authors: Cheng Dong, Heng Zhang, Wolfram Tempel, Peter Loppnau, Jinrong MinAbstract:The N-end rule pathway senses the N-terminal destabilizing residues of degradation substrates for the ubiquitin-proteasome system, whose integrity shields against various human syndromes including cancer and cardiovascular diseases. GID4, a subunit of the ubiquitin ligase GID complex, has been recently identified as the N-recognin of the new branch of the N-end rule pathway responsible for recognizing substrates bearing N-terminal proline residues (Pro/N-Degrons). However, the molecular mechanism of GID4-mediated Pro/N-Degron recognition remains largely unexplored. Here, we report the first crystal structures of human GID4 alone and in complex with various Pro/N-Degrons. Our complex crystal structures, together with biophysical analyses, delineate the GID4-mediated Pro/N-Degron recognition mechanism and substrate selection criteria for the Pro/N-end rule pathway. These mechanistic data on the Pro/N-recognin activity of GID4 will serve as a foundation to facilitate the identification of authentic physiological substrates as well as the development of inhibitors of therapeutic values for the Pro/N-end rule pathway.
Nico Dissmeyer - One of the best experts on this subject based on the ideXlab platform.
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conditional protein function via n Degron pathway mediated proteostasis in stress physiology
Annual Review of Plant Biology, 2019Co-Authors: Nico DissmeyerAbstract:The N-Degron pathway, formerly the N-end rule pathway, regulates functions of regulatory proteins. It impacts protein half-life and therefore directs the actual presence of target proteins in the cell. The current concept holds that the N-Degron pathway depends on the identity of the amino (N)-terminal amino acid and many other factors, such as the follow-up sequence at the N terminus, conformation, flexibility, and protein localization. It is evolutionarily conserved throughout the kingdoms. One possible entry point for substrates of the N-Degron pathway is oxidation of N-terminal Cys residues. Oxidation of N-terminal Cys is decisive for further enzymatic modification of various neo-N termini by arginylation that generates potentially neofunctionalized or instable proteoforms. Here, I focus on the posttranslational modifications that are encompassed by protein degradation via the Cys/Arg branch of the N-Degron pathway-part of the PROTEOLYSIS 6 (PRT6)/N-Degron pathway-as well as the underlying physiological principles of this branch and its biological significance in stress response.
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Conditional Protein Function via N-Degron Pathway–Mediated Proteostasis in Stress Physiology
Annual review of plant biology, 2019Co-Authors: Nico DissmeyerAbstract:The N-Degron pathway, formerly the N-end rule pathway, regulates functions of regulatory proteins. It impacts protein half-life and therefore directs the actual presence of target proteins in the cell. The current concept holds that the N-Degron pathway depends on the identity of the amino (N)-terminal amino acid and many other factors, such as the follow-up sequence at the N terminus, conformation, flexibility, and protein localization. It is evolutionarily conserved throughout the kingdoms. One possible entry point for substrates of the N-Degron pathway is oxidation of N-terminal Cys residues. Oxidation of N-terminal Cys is decisive for further enzymatic modification of various neo-N termini by arginylation that generates potentially neofunctionalized or instable proteoforms. Here, I focus on the posttranslational modifications that are encompassed by protein degradation via the Cys/Arg branch of the N-Degron pathway-part of the PROTEOLYSIS 6 (PRT6)/N-Degron pathway-as well as the underlying physiological principles of this branch and its biological significance in stress response.
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Conditional Modulation of Biological Processes by Low-Temperature Degrons.
Methods in molecular biology (Clifton N.J.), 2017Co-Authors: Nico DissmeyerAbstract:Conditional modulation of biological processes plays key roles in basic and applied research and in translation. It can be achieved on various levels via a multitude of approaches. One of the directions is manipulating target protein levels and activity by transcriptional, posttranscriptional, translational, and posttranslational control. Because in most of these techniques, the synthesis of the target proteins is adjusted to the needs, they all rely on the specific half-life of the target protein and its turn-over. Therefore, their time-of-action, in direct correlation to the desired reprogramming of molecular phenotypes caused by altering the target levels, is fixed and determined by the naturally inherent properties. We have introduced the low-temperature Degron (lt-Degron) to various intact multicellular organisms which allows to control target protein levels and therefore function and activity directly on the level of active protein. The lt-Degron uses a combination of Ubiquitin-fusion technique linking target protein degradation to the N-end rule pathway of targeted proteolysis coupled with the use of cell- and tissue-specific promoters.
Cheng Dong - One of the best experts on this subject based on the ideXlab platform.
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Molecular basis for ubiquitin ligase CRL2FEM1C-mediated recognition of C-Degron
Nature chemical biology, 2021Co-Authors: Xiaojie Yan, Xiaolu Wang, Mengqi Zhou, Lili Song, Jinrong Min, Cheng DongAbstract:Proteome integrity depends on the ubiquitin-proteasome system to degrade unwanted or abnormal proteins. In addition to the N-Degrons, C-terminal residues of proteins can also serve as degradation signals (C-Degrons) that are recognized by specific cullin-RING ubiquitin ligases (CRLs) for proteasomal degradation. FEM1C is a CRL2 substrate receptor that targets the C-terminal arginine Degron (Arg/C-Degron), but the molecular mechanism of substrate recognition remains largely elusive. Here, we present crystal structures of FEM1C in complex with Arg/C-Degron and show that FEM1C utilizes a semi-open binding pocket to capture the C-terminal arginine and that the extreme C-terminal arginine is the major structural determinant in recognition by FEM1C. Together with biochemical and mutagenesis studies, we provide a framework for understanding molecular recognition of the Arg/C-Degron by the FEM family of proteins.
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Recognition of nonproline N-terminal residues by the Pro/N-Degron pathway
Proceedings of the National Academy of Sciences of the United States of America, 2020Co-Authors: Cheng Dong, Alexander Varshavsky, Artem Melnykov, Shun-jia Chen, Sara Weirich, Kelly Sun, Albert Jeltsch, Jinrong MinAbstract:Eukaryotic N-Degron pathways are proteolytic systems whose unifying feature is their ability to recognize proteins containing N-terminal (Nt) degradation signals called N-Degrons, and to target these proteins for degradation by the 26S proteasome or autophagy. GID4, a subunit of the GID ubiquitin ligase, is the main recognition component of the proline (Pro)/N-Degron pathway. GID4 targets proteins through their Nt-Pro residue or a Pro at position 2, in the presence of specific downstream sequence motifs. Here we show that human GID4 can also recognize hydrophobic Nt-residues other than Pro. One example is the sequence Nt-IGLW, bearing Nt-Ile. Nt-IGLW binds to wild-type human GID4 with a K d of 16 μM, whereas the otherwise identical Nt-Pro-bearing sequence PGLW binds to GID4 more tightly, with a K d of 1.9 μM. Despite this difference in affinities of GID4 for Nt-IGLW vs. Nt-PGLW, we found that the GID4-mediated Pro/N-Degron pathway of the yeast Saccharomyces cerevisiae can target an Nt-IGLW-bearing protein for rapid degradation. We solved crystal structures of human GID4 bound to a peptide bearing Nt-Ile or Nt-Val. We also altered specific residues of human GID4 and measured the affinities of resulting mutant GID4s for Nt-IGLW and Nt-PGLW, thereby determining relative contributions of specific GID4 residues to the GID4-mediated recognition of Nt-Pro vs. Nt-residues other than Pro. These and related results advance the understanding of targeting by the Pro/N-Degron pathway and greatly expand the substrate recognition range of the GID ubiquitin ligase in both human and yeast cells.
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recognition of nonproline n terminal residues by the pro n Degron pathway
Proceedings of the National Academy of Sciences of the United States of America, 2020Co-Authors: Cheng Dong, Alexander Varshavsky, Jinrong Min, Artem Melnykov, Shun-jia Chen, Sara Weirich, Kelly Sun, Albert JeltschAbstract:Eukaryotic N-Degron pathways are proteolytic systems whose unifying feature is their ability to recognize proteins containing N-terminal (Nt) degradation signals called N-Degrons, and to target these proteins for degradation by the 26S proteasome or autophagy. GID4, a subunit of the GID ubiquitin ligase, is the main recognition component of the proline (Pro)/N-Degron pathway. GID4 targets proteins through their Nt-Pro residue or a Pro at position 2, in the presence of specific downstream sequence motifs. Here we show that human GID4 can also recognize hydrophobic Nt-residues other than Pro. One example is the sequence Nt-IGLW, bearing Nt-Ile. Nt-IGLW binds to wild-type human GID4 with a K d of 16 μM, whereas the otherwise identical Nt-Pro-bearing sequence PGLW binds to GID4 more tightly, with a K d of 1.9 μM. Despite this difference in affinities of GID4 for Nt-IGLW vs. Nt-PGLW, we found that the GID4-mediated Pro/N-Degron pathway of the yeast Saccharomyces cerevisiae can target an Nt-IGLW-bearing protein for rapid degradation. We solved crystal structures of human GID4 bound to a peptide bearing Nt-Ile or Nt-Val. We also altered specific residues of human GID4 and measured the affinities of resulting mutant GID4s for Nt-IGLW and Nt-PGLW, thereby determining relative contributions of specific GID4 residues to the GID4-mediated recognition of Nt-Pro vs. Nt-residues other than Pro. These and related results advance the understanding of targeting by the Pro/N-Degron pathway and greatly expand the substrate recognition range of the GID ubiquitin ligase in both human and yeast cells.
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Molecular basis of GID4-mediated recognition of Degrons for the Pro/N-end rule pathway.
Nature chemical biology, 2018Co-Authors: Cheng Dong, Heng Zhang, Wolfram Tempel, Peter Loppnau, Jinrong MinAbstract:The N-end rule pathway senses the N-terminal destabilizing residues of degradation substrates for the ubiquitin-proteasome system, whose integrity shields against various human syndromes including cancer and cardiovascular diseases. GID4, a subunit of the ubiquitin ligase GID complex, has been recently identified as the N-recognin of the new branch of the N-end rule pathway responsible for recognizing substrates bearing N-terminal proline residues (Pro/N-Degrons). However, the molecular mechanism of GID4-mediated Pro/N-Degron recognition remains largely unexplored. Here, we report the first crystal structures of human GID4 alone and in complex with various Pro/N-Degrons. Our complex crystal structures, together with biophysical analyses, delineate the GID4-mediated Pro/N-Degron recognition mechanism and substrate selection criteria for the Pro/N-end rule pathway. These mechanistic data on the Pro/N-recognin activity of GID4 will serve as a foundation to facilitate the identification of authentic physiological substrates as well as the development of inhibitors of therapeutic values for the Pro/N-end rule pathway.