Means of self-preservation: how an intrinsically disordered ubiquitin-protein ligase averts self-destruction

Fredrickson, Eric K.; Candadai, Sarah V. Clowes; Tam, Cheuk Ho; Gardner, Richard G.

doi:10.1091/mbc.e12-11-0811

Cited by 27 publications

(35 citation statements)

References 30 publications

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“…The protein interaction database (16,(33)(34)(35)(36) revealed the interaction of San1 with the Spt16 component of FACT that is involved in promoting transcriptional elongation (5,6). On the basis of this information, we hypothesize that San1 regulates transcriptional elongation, possibly by controlling the stability or abundance of Spt16 via ubiquitylation and proteasomal degradation.…”

Section: Resultsmentioning

confidence: 94%

Fine-Tuning of FACT by the Ubiquitin Proteasome System in Regulation of Transcriptional Elongation

Sen

Ferdoush

Kaja

et al. 2016

Molecular and Cellular Biology

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FACT (facilitates chromatin transcription), an evolutionarily conserved histone chaperone involved in transcription and other DNA transactions, is upregulated in cancers, and its downregulation is associated with cellular death. However, it is not clearly understood how FACT is fine-tuned for normal cellular functions. Here, we show that the FACT subunit Spt16 is ubiquitylated by San1 (an E3 ubiquitin ligase) and degraded by the 26S proteasome. Enhanced abundance of Spt16 in the absence of San1 impairs transcriptional elongation. Likewise, decreased abundance of Spt16 also reduces transcription. Thus, an optimal level of Spt16 is required for efficient transcriptional elongation, which is maintained by San1 via ubiquitylation and proteasomal degradation. Consistently, San1 associates with the coding sequences of active genes to regulate Spt16's abundance. Further, we found that enhanced abundance of Spt16 in the absence of San1 impairs chromatin reassembly at the coding sequence, similarly to the results seen following inactivation of Spt16. Efficient chromatin reassembly enhances the fidelity of transcriptional elongation. Taken together, our results demonstrate for the first time a fine-tuning of FACT by a ubiquitin proteasome system in promoting chromatin reassembly in the wake of elongating RNA polymerase II and transcriptional elongation, thus revealing novel regulatory mechanisms of gene expression. In eukaryotes, DNA is packaged into nucleosomes to form chromatin. Each nucleosome within chromatin consists of ϳ147 bp of DNA wrapped around a histone octamer containing one histone H3-H4 tetramer and two histone H2A-H2B dimers (1). Thus, chromatin structure plays crucial functions in regulation of DNA transactions such as transcription, replication, and DNA repair (2-4). A variety of factors are involved in altering chromatin structure and, hence, DNA transactions. These factors are generally classified as ATP-independent histone modifying enzymes, ATP-dependent chromatin remodelers, and histone chaperones. Histone modifying enzymes function through addition or removal of specific chemical groups (e.g., acetyl, methyl, ubiquitin, phospho, and SUMO), while ATP-dependent chromatin remodelers alter DNA-histone interactions or the composition of the nucleosome in an ATP-dependent manner. On the other hand, histone chaperones function by binding with nucleosomes or histones to facilitate assembly and/or disassembly of nucleosomes in an ATP-independent fashion. The histone chaperone that was first found to alter chromatin structure during transcription is FACT (facilitates chromatin transcription), which is evolutionarily conserved among eukaryotes (5, 6). In budding yeast (Saccharomyces cerevisiae), FACT is composed of Spt16 (suppressor of Ty) and Pob3 and physically interacts with nucleosomes with the assistance of the HMG (high mobility group) protein Nhp6. Likewise, FACT is also a heterodimer of Spt16 and SSRP1 (structurespecific recognition protein 1) in humans. SSRP1 contains HMG domain, while HMG domain is p...

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Section: Resultsmentioning

confidence: 94%

Fine-Tuning of FACT by the Ubiquitin Proteasome System in Regulation of Transcriptional Elongation

Sen

Ferdoush

Kaja

et al. 2016

Molecular and Cellular Biology

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“…1). Because a single lysine San1 would allow for the specific monitoring of polyubiquitin chain formation without the potentially confounding effects of multi-monoubiquitylation, all naturally occurring San1 lysine residues were mutated to arginine, and a single lysine was introduced at Asn-13 because previous experiments in yeast demonstrated that N13K San1 was both autoubiquitylated and capable of binding to misfolded proteins (38).…”

Section: Resultsmentioning

confidence: 99%

“…It had previously been shown that introducing a small, hydrophobic patch in the San1 amino acid sequence resulted in increased turnover of San1 protein in yeast (38). In those experiments, the RING domain in San1 had been mutated to eliminate autoubiquitylation.…”

Section: Resultsmentioning

confidence: 99%

“…Cloning-All San1 constructs were amplified by PCR from previously published templates (38). The 5Ј primer contained coding sequences for a tobacco etch virus protease cleavage site followed by a protein kinase A (PKA) phosphorylation consensus motif (5Ј-GGCGGATCCGAGAACCTGTACTTCCAG-GGCCGTCGCGGTAGCCTGAGTGAAAGTGGTCAAGAA-CAAAACA-3Ј), and the 3Ј primer contained a coding sequence for an eight-histidine tag that was appended to the San1 C terminus (5Ј-GGCCTCGAGTTAATGGTGATGGTGATGG-TGATGGTGTTGTGATGATCGTTGCTCATTG-3Ј).…”

Section: Methodsmentioning

confidence: 99%

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The San1 Ubiquitin Ligase Functions Preferentially with Ubiquitin-conjugating Enzyme Ubc1 during Protein Quality Control

Ibarra

Sandoval

Fredrickson

et al. 2016

Journal of Biological Chemistry

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Protein quality control (PQC) is a critical process wherein misfolded or damaged proteins are cleared from the cell to maintain protein homeostasis. In eukaryotic cells, the removal of misfolded proteins is primarily accomplished by the ubiquitin-proteasome system. In the ubiquitin-proteasome system, ubiquitin-conjugating enzymes and ubiquitin ligases append polyubiquitin chains onto misfolded protein substrates signaling for their degradation. The kinetics of protein ubiquitylation are paramount as a balance must be achieved between the rapid removal of misfolded proteins versus providing sufficient time for protein chaperones to attempt refolding. To uncover the molecular basis for how PQC substrate ubiquitylation rates are controlled, the reaction catalyzed by nuclear ubiquitin ligase San1 was reconstituted in vitro. Our results demonstrate that San1 can function with two ubiquitin-conjugating enzymes, Cdc34 and Ubc1. Although Cdc34 and Ubc1 are both sufficient for promoting San1 activity, San1 functions preferentially with Ubc1, including when both Ubc1 and Cdc34 are present. Notably, a homogeneous peptide that mimics a misfolded PQC substrate was developed and enabled quantification of the kinetics of San1-catalyzed ubiquitylation reactions. We discuss how these results may have broad implications for the regulation of PQC-mediated protein degradation.For cells to maintain proteostasis, a delicate balance must exist between protein biogenesis and degradation. The ubiquitin-proteasome system is a network of proteins and enzymes responsible for 70 -80% of intracellular protein degradation in eukaryotic cells (1). The signal for protein degradation is the assembly of a polyubiquitin chain onto protein substrate.Ubiquitylation occurs through the sequential action of three enzymes: E1 2 (ubiquitin-activating enzyme), E2 (ubiquitinconjugating enzyme), and E3 (ubiquitin ligase) (2-5). E1 activates ubiquitin, a highly conserved 76-amino acid protein forming a thioester bond between the C-terminal carboxyl group on ubiquitin and a cysteine residue located within the E1 active site. Next, ubiquitin is transferred from E1 to E2. The E2ϳubiquitin (ϳ is used to denote a thioester bond) is then recruited by an E3, which brings the E2ϳubiquitin and protein substrate into proximity. E3s may also participate in ubiquitylation by stimulating the ubiquitin transfer activity of E2s (6 -11). In most cases ubiquitin is transferred from E2ϳubiquitin to the protein substrate, forming an isopeptide bond between the ubiquitin C terminus and a lysine residue on the substrate. The dissociation of E2s from E3 and the binding of fresh E2ϳubiquitin complexes enables the formation of a polyubiquitin chain on the substrate. Typically, a chain of at least four ubiquitins is required for a substrate to be recognized by the 26S proteasome for degradation (12, 13); however, the modification of several lysine residues with a single ubiquitin on some substrates may also be sufficient (14, 15). PQC is a collection of critical pathways within the ...

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“…San1’s ability to target misfolded nuclear proteins is attributed to large regions of intrinsic disorder within the N-and C-terminal regions of San1 [12]. These disordered regions possess multiple substrate-interaction regions within a conformationally plastic scaffold, which we propose provides San1 a broad mechanism to target many different kinds of misfolded proteins [12,35]. San1 has been found to recognize exposed hydrophobicity within its misfolded substrates that would have normally been buried within the protein [11].…”

Section: Pqc Degradation In the Nucleusmentioning

confidence: 99%

Protein quality control in the nucleus

Jones

Gardner

2016

Current Opinion in Cell Biology

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The nucleus is the repository for the eukaryotic cell’s genetic blueprint, which must be protected from harm to ensure survival. Multiple quality control (QC) pathways operate in the nucleus to maintain the integrity of the DNA, the fidelity of the DNA code during replication, its transcription into mRNA, and the functional structure of the proteins that are required for DNA maintenance, mRNA transcription, and other important nuclear processes. Although we understand a great deal about DNA and RNA QC mechanisms, we know far less about nuclear protein quality control (PQC) mechanisms despite that fact that many human diseases are causally linked to protein misfolding in the nucleus. In this review, we discuss what is known about nuclear PQC and we highlight new questions that have emerged from recent developments in nuclear PQC studies.

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Means of self-preservation: how an intrinsically disordered ubiquitin-protein ligase averts self-destruction

Cited by 27 publications

References 30 publications

Fine-Tuning of FACT by the Ubiquitin Proteasome System in Regulation of Transcriptional Elongation

Fine-Tuning of FACT by the Ubiquitin Proteasome System in Regulation of Transcriptional Elongation

The San1 Ubiquitin Ligase Functions Preferentially with Ubiquitin-conjugating Enzyme Ubc1 during Protein Quality Control

Protein quality control in the nucleus

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