Sequence- and Species-Dependence of Proteasomal Processivity

Abstract

The proteasome is the degradation machine at the center of the ubiquitin-proteasome system and controls the concentrations of many proteins in eukaryotes. It is highly processive so that substrates are degraded completely into small peptides, avoiding the formation of potentially toxic fragments. Nonetheless, some proteins are incompletely degraded, indicating the existence of factors that influence proteasomal processivity. We have quantified proteasomal processivity and determined the underlying rates of substrate degradation and release. We find that processivity increases with species complexity over a 5-fold range between yeast and mammalian proteasome, and the effect is due to slower but more persistent degradation by proteasomes from more complex organisms. A sequence stretch that has been implicated in causing incomplete degradation, the glycine-rich region of the NFκB subunit p105, reduces the proteasome’s ability to unfold its substrate, and polyglutamine repeats such as found in Huntington’s disease reduce the processivity of the proteasome in a length-dependent manner.

Giant axonal neuropathy–associated gigaxonin mutations impair intermediate filament protein degradation

“…76); pET30A-WT gigaxonin. As a control, we used a construct encoding sic 60-barnase-DHFR fusion -consisting of 60 aas from the bacterial Sic1 protein (containing a PPXY Rsp5 ubiquitination motif); followed by a catalytically inactive (H102A) Bacillus amyloliquefaciens barnase, with all lysine replaced by arginine, methionine, or alanine residues; followed by E. coli dihydrofolate reductase (DHFR) -that has previously been used for ubiquitination and unfolding studies (77). Radioactive vimentin and control substrates (in pGEM3zf[+]) were expressed from a T7 promoter by in vitro transcription and translation using a TNT Coupled Reticulocyte Lysate system (Promega) or an E. coli T7 S30 Extract system (Promega) containing [ 35 S]-methionine.…”

“…The ubiquitination reaction mixture contained 100 nM UbE1, 200 ng UbcH5a (E2), 1 mg/ml ubiquitin, and 2 μM ubiquitin aldehyde (all from Boston Biochem); 20 mM creatine phosphate and 0.2 mg/ml creatine phosphokinase (both from Roche); 4 mM ATP (Sigma-Aldrich); and 1 μM DTT in ubiquitination buffer (25 mM Tris-Cl, pH 7.5; 50 mM NaCl; and 4 mM MgCl2), as described previously (78). In addition, in vitro-transcribed and -translated gigaxonin and Cullin-3 (supplemented with nonradioactive methionine) or Rsp5 (E3) were added to assay for the ubiquitination of vimentin or the control substrates (77,78). The reaction was incubated at 30°C for 90 minutes, and then samples were placed in Laemmli sample buffer and analyzed by SDS-PAGE.…”

The E3 Ubiquitin Ligase UBE3C Enhances Proteasome Processivity by Ubiquitinating Partially Proteolyzed Substrates

“…To address this question, we created a cell line expressing DD fused to wild-type E. coli DHFR, a protein that becomes more stably folded upon binding to the small molecule methotrexate (MTX) (18,44). Knockdown of UBE3C in this cell line had no effect on fragment formation in this cell line as we observed fragment formation upon ligand removal was dependent solely upon the presence or absence of MTX (Fig.…”

Slippery Substrates Impair ATP-dependent Protease Function by Slowing Unfolding

“…These proteases mostly recognize their substrates through sequence motifs at the N or C termini of the substrate proteins, and different proteases appear to have overlapping specificities (1)(2)(3). Additionally, these proteases appear to differ in their intrinsic ability to unfold and degrade substrate proteins, which may also be substrate-dependent, perhaps providing an additional contribution to the specificity of protein degradation (3)(4)(5)(6).…”

“…The GRR sequence reduces the processivity of the proteasome in its normal biological context (p105), when combined with proteins such as dihydrofolate reductase (DHFR) in in vitro degradation assays, and also reduces the processivity of bacterial proteases such as ClpXP and ClpAP (3,6,14). Another low complexity sequence, a Gly-Ala repeat (GAr) in the EBNA1 (Epstein-Barr virus nuclear antigen-1) protein, inhibits proteasomal degradation of EBNA1 in cells, reduces proteasomal processivity in model substrates, and has recently been shown to reduce processivity of the bacterial protease ClpXP (15)(16)(17)(18)(19)(20).…”