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Defining the geometry of the two-component proteasome degron.

ABSTRACT: The eukaryotic 26S proteasome controls cellular processes by degrading specific regulatory proteins. Most proteins are targeted for degradation by a signal or degron that consists of two parts: a proteasome-binding tag, typically covalently attached polyubiquitin chains, and an unstructured region that serves as the initiation region for proteasomal proteolysis. Here we have characterized how the arrangement of the two degron parts in a protein affects degradation. We found that a substrate is degraded efficiently only when its initiation region is of a certain minimal length and is appropriately separated in space from the proteasome-binding tag. Regions that are located too close or too far from the proteasome-binding tag cannot access the proteasome and induce degradation. These spacing requirements are different for a polyubiquitin chain and a ubiquitin-like domain. Thus, the arrangement and location of the proteasome initiation region affect a protein's fate and are important in selecting proteins for proteasome-mediated degradation.

SUBMITTER: Inobe T

PROVIDER: S-EPMC3129032 | biostudies-literature | 2011 Mar

REPOSITORIES: biostudies-literature

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Defining the geometry of the two-component proteasome degron.

Inobe Tomonao T Fishbain Susan S Prakash Sumit S Matouschek Andreas A

Nature chemical biology 20110130 3

The eukaryotic 26S proteasome controls cellular processes by degrading specific regulatory proteins. Most proteins are targeted for degradation by a signal or degron that consists of two parts: a proteasome-binding tag, typically covalently attached polyubiquitin chains, and an unstructured region that serves as the initiation region for proteasomal proteolysis. Here we have characterized how the arrangement of the two degron parts in a protein affects degradation. We found that a substrate is d ...[more]

PMID: 21278740

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Project description:BACKGROUND: We identified that a putative CzcRS-like two-component system (TCS) in Burkholderia cenocepacia K56-2 is required for heavy metal resistance and virulence. To investigate this TCS further, we applied RNA-seq to determine the transcriptional response of wildtype and CzcRS-deficient B. cenocepacia to subinhibitory concetrations of zinc. METHODS. Wildtype B. cenocepacia K56-2 was cultured in LB broth with and without 1.5 mM zinc chloride. In parallel, a ΔczcRS mutant strain was also cultured in 1.5 mM zinc chloride. Total RNA was extracted from triplicate mid log-phase cultures for each strain/culture condition. Illumina Sequencing libraries were prepared from 50 ng ribosomal-depleted RNA using ScriptSeq v2 with indexing and 15 cycles of PCR amplification using Kapa HiFi DNA polymerase. The amplified libraries were purified using a 1:1 ratio of Ampure XP beads resulting in 45-75nM concentrations and average insert size of 350 bp. Libraries were pooled in equimolar concentrations, denatured and diluted to 6.5 pM before clustering on a cBOT (Illumina). Libraries were subjected to 100-bp paired-end sequencing on a HiSeq 2000 using TruSeq SBS v 3 reagents (Illumina). Paired-end reads were filtered using the fastq-mcf package from the ea-utils suite to remove reads with less than 90% Q20 scores or above and to trim off adaptor sequence. The reads were then aligned against the B. cenocepacia J2315 genome (NC_011001-NC_011003) using Bowtie 1 to remove reads mapping to ERCC spike-in. Abundance of ERCC spike-ins was then checked against predicted values to confirm successful library preparation. Tophat v2.0.4 was used to align reads against the reference genome. The Bedtools multicov command was used to extract per-gene counts for each gene. The DESeq suite was used to determine statistical significance of differential expression between conditions. RESULTS: Wildtype B. cenocepacia K56-2 showed a restricted transcriptional response to 1.5 mM zinc chloride, with only 54 genes exhibiting significant differential expression (≥ 1.5-fold; P < 0.05) compared to unsupplemented LB. This transcriptional response included genes encoding the CzcRS-like TCS itself and the associated efflux pump (CzcCBA). In contrast, the ΔczcRS mutant strain when cultured in 1.5 mM zinc chloride exhbited a profound transcriptional response with 767 genes differentially expressed. Many genes showing differential expression in the ΔczcRS strain are indicative of a stress response, indicating the pivotal role of the CzcRS-like TCS in maintaining cellular homeostasis in response to zinc.

Project description:BACKGROUND: We identified that a putative CzcRS-like two-component system (TCS) in Burkholderia cenocepacia K56-2 is required for heavy metal resistance and virulence. In an attempt to identify genes directly regulated by the CzcR response regulator, we performed ChIP-seq analysis to identify genomic regions bound by CzcR. METHODS. Sequence encoding a FLAG octapeptide (Sigma-Aldrich) was introduced to the 3’ end of the czcR gene at its native chromosomal position in wild-type B. cenocepacia K56-2. Wildtype K56-2 and the CzcR-FLAG strain were grown in 50 ml tryptone soya broth (TSB) in the presence or absence of 1.5 mM zinc chloride. After overnight incubation, anti-FLAG immunoprecipitation of CzcR-bound genomic DNA was performed. Wildtype K56-2 was processed in parallel, to serve as a control against which to assess for enrichment of genomic regions. Illumina sequencing libraries were prepared from the resulting DNA using the Nextflex ChIPseq protocol (Bioo Scientific) with indexed adapters. Libraries were amplified by 18 cycles PCR, purified using 0.8 volumes Ampure XP beads (Beckman Coulter) and quantified with a Bioanalyzer 7500 assay (Agilent). Libraries ranged in size from 150 bp to 800 bp with a average insert size of 310 bp. Libraries were pooled in equimolar concentrations, denatured and diluted to 6.5 pM, clustered on a flowcell using a cBOT (Illumina) and sequenced on a HiSeq2000 (Illumina). Paired-end reads were filtered using the fastq-mcf package from the ea-utils suite to remove reads with less than 90% Q20 scores or above and to trim off adaptor sequence. The reads were then aligned against the B. cenocepacia J2315 genome (NC_011001-NC_011003) using BWA (0.5.9) and converted to BAM format using Samtools. Potential PCR duplicates were removed using the samtools rmdup command. The MACS package (v1.4) was used to compare and contrast the control and sample data using the –call-subpeaks and –w options. RESULTS: Relative to wildtype B. cenocepacia K56-2, the only genomic region that was found to be enriched by the ChIP-seq analysis of the CzcR-FLAG strain mapped to nucleotide coordinates 787809-798988 on chromosome 2. This region includes the czcRS genes and those encoding the associated efflux pump (CzcCBA).

Dataset Information

Defining the geometry of the two-component proteasome degron.

Publications

Defining the geometry of the two-component proteasome degron.

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