Immunoproteasome functions explained by divergence in cleavage specificity and regulation

  1. Michael B Winter
  2. Florencia La Greca
  3. Shirin Arastu-Kapur
  4. Francesco Caiazza
  5. Peter Cimermancic
  6. Tonia J Buchholz
  7. Janet L Anderl
  8. Matthew Ravalin
  9. Markus F Bohn
  10. Andrej Sali
  11. Anthony J O'Donoghue  Is a corresponding author
  12. Charles S Craik  Is a corresponding author
  1. University of California, San Francisco, United States
  2. Inc., an Amgen subsidiary, United States
  3. California Institute for Quantitative Biosciences, University of California, San Francisco, United States
  4. University of California, San Diego, United States
5 figures and 4 additional files

Figures

Figure 1 with 4 supplements
Global substrate specificity profiling of the iP and cP with Multiplex Substrate Profiling by Mass Spectrometry (MSP-MS) reveals shared and differential substrate specificity features.

(A) iceLogo representations of iP and cP substrate specificity (P4–P4ʹ) at the 480 min assay time point (p≤0.05 for non-grayed residues (Colaert et al., 2009); ‘n’ is norleucine). (B) Quantification …

https://doi.org/10.7554/eLife.27364.002
Figure 1—source data 1

The following are contained in a supplementary file for the MSP-MS assay: a sample key; the sequences of the peptide library in FASTA format; a full mass spectrometry peptide report; processed cleavage data showing spectral counts for each octapeptide (P4–P4ʹ) and the associated cleavage within the parental peptide; processed cleavage data (indicated as ‘negative’) with cleavages also appearing in the no-enzyme control (NEC) highlighted; all octapeptides (P4–P4ʹ) to be used as the background dataset in iceLogo; ‘positive’ octapeptide (P4–P4ʹ) cleavages with the ‘negative’ cleavage data removed; ‘accumulative’ octapeptide (P4–P4ʹ) cleavages at each assay time point for the proteasome biological replicates; a comparison of cleavages common between biological replicates; and heat maps based on Z-scores showing the specificity at each assay time point.

https://doi.org/10.7554/eLife.27364.007
Figure 1—figure supplement 1
Comparison of iP- and cP-derived peptide cleavage products across all time points in the MSP-MS assay.

(A) Progress curves for the iP and cP are shown demonstrating the kinetics of peptide cleavage during the MSP-MS assay time course. (B) Quantification of shared and non-overlapping cleavages is …

https://doi.org/10.7554/eLife.27364.003
Figure 1—figure supplement 2
The MSP-MS assay detects the most dominant peptide cleavage events.

These cleavage events are those that tend to occur initially and produce peptides of appropriate size (in m/z). For a given protease, the assay is capable of detecting up to approximately 20% of the …

https://doi.org/10.7554/eLife.27364.004
Figure 1—figure supplement 3
Heat maps for the iP and cP across multiple MSP-MS assay time points demonstrating shared and differential specificity features at the P4 to P4ʹ positions.

The heat map from the 480 min assay time point has been repeated from Figure 1 for clarity.

https://doi.org/10.7554/eLife.27364.005
Figure 1—figure supplement 4
Qualitative comparison of the major differences in human iP and cP P1 specificity preferences identified using the MSP-MS library compared to those reported in previous profiling studies.

Toes et al used a model protein substrate (enolase-1) for proteasome cleavage, (Toes et al., 2001) whereas Mishto et al used a set of synthetic peptides corresponding to the sequences gp10035-57, …

https://doi.org/10.7554/eLife.27364.006
Figure 2 with 4 supplements
Rational optimization of tetrapeptide ACC substrates with iP and cP selectivity.

(A) Example 14-mer peptides from the iP and cP MSP-MS assays associated with Figure 1 illustrating cleavages that were used as a template for rational fluorogenic substrate design. All cleavages …

https://doi.org/10.7554/eLife.27364.008
Figure 2—figure supplement 1
P4-P2 substrate specificity of SDS-activated iP and cP using the PS-SCL profiling approach.

Mean activity is reported with error bars representing the standard deviation from n = 3 replicates.

https://doi.org/10.7554/eLife.27364.009
Figure 2—figure supplement 2
PS-SCL profiling of SDS-activated iP and cP in the absence and presence of 40 nM CFZ pretreatment.

CFZ levels were selected based on inhibitor titration against the library to achieve optimal selectivity. Mean activity is reported with error bars representing the standard deviation from n = 3 …

https://doi.org/10.7554/eLife.27364.010
Figure 2—figure supplement 3
Synthetic approach for peptide substrates bearing a C-terminal ACC fluorophore.

Protocols for synthesis of the Fmoc-ACC fluorophore and its coupling to Rink amide resin have been described. (Maly et al., 2002) (a) 20% 4-methylpiperidine/DMF; (b) Fmoc-protected P1 amino acid (5 …

https://doi.org/10.7554/eLife.27364.011
Figure 2—figure supplement 4
Comparison of fluorogenic substrate selectivity under SDS (0.03%) and PA28 (12 eq.) activation conditions.

Mean activity is reported with error bars representing the standard deviation from n = 3 replicates.

https://doi.org/10.7554/eLife.27364.012
Figure 3 with 3 supplements
Optimal fluorogenic substrates enable the selective monitoring of LMP7 and β5 activity in cell lysates containing varied proteasome ratios.

(A) Demonstration of EWFW-ACC (10 μM) and iso-VQA-ACC (30 μM) selectivity for the iP and cP, respectively, compared to the universal substrate LLVY-AMC (30 μM) using mixed ratios of purified …

https://doi.org/10.7554/eLife.27364.013
Figure 3—figure supplement 1
Michaelis-Menten plot of the iP and cP with the commercial LLVY-AMC substrate.

Km = 98.6 μM for the iP and 65.7 μM for the cP with 2.0 fold selectivity at Vmax.

https://doi.org/10.7554/eLife.27364.014
Figure 3—figure supplement 2
Quantification of active LMP7 and β5 levels in lysates from whole blood, the MOLT-4 cell line, and PBMCs through direct active site labeling using the ProCISE assay.

Mean labeled subunit levels are reported with error bars representing the standard deviation from n = 3 replicates.

https://doi.org/10.7554/eLife.27364.015
Figure 3—figure supplement 3
Improved ability of the iso-VQA-ACC substrate to detect β5 activity in the MOLT-4 lysate compared to a commercially available β5 fluorogenic substrate.

Mean activity is reported with error bars representing the standard deviation from n = 3 replicates.

https://doi.org/10.7554/eLife.27364.016
Figure 4 with 2 supplements
Differential substrate specificity of the iP alters MHC I peptide cleavage and is likely due to neutral evolution from the ancestral cP.

(A) Label-free quantitation of relative iP and cP cleavage rates against a library of synthetic peptides derived from sequences flanking sites of MHC I peptide processing (Bassani-Sternberg et al., …

https://doi.org/10.7554/eLife.27364.017
Figure 4—source data 1

The following are contained in a supplementary file for the MHC cleavage assay: a sample key; the sequences of the peptide library in FASTA format; and a full mass spectrometry peptide report.

https://doi.org/10.7554/eLife.27364.018
Figure 4—figure supplement 1
Prediction of MHC I peptide cleavage by the iP and cP.

(A) Scoring of the MHC I peptidome (Bassani-Sternberg et al., 2015) (n = 22,598) based on the relative favorability for cleavage by the iP or cP. Scores were derived from the P4-P1 cleavage …

https://doi.org/10.7554/eLife.27364.019
Figure 4—figure supplement 2
Protein sequence alignment for (A) β5 and (B) LMP7 in diverse species containing both proteasomes.

(Sutoh et al., 2012) UniProt identification numbers are respectively as follows for β5 and LMP7: P28074 and P28062, Homo sapiens (human); O55234 and P28063, Mus musculus (mouse); P28075 and P28064, R…

https://doi.org/10.7554/eLife.27364.020
Figure 5 with 3 supplements
iP activity makes a significant contribution to UPS capacity and undergoes selective and coordinated recovery from inhibition.

(A) Ubiquitin western blot and matched activity assay for the SUP-B15 cell line (with approximately 80:20 LMP7: β5 activity) showing a substantial increase in global ubiquitination levels upon …

https://doi.org/10.7554/eLife.27364.021
Figure 5—figure supplement 1
Quantification of the ubiquitin levels in the SUP-B15 cell line at the one- and four-hour time points following one-hour pulse treatment with ONX 0914 and PR-825 (Figure 5A).

Mean ubiquitin levels are reported with normalization to the untreated condition. Statistically significant differences compared to the untreated condition are indicated (*p≤0.05) as determined with …

https://doi.org/10.7554/eLife.27364.022
Figure 5—figure supplement 2
Recovery of LMP7 activity in PBMCs from n = 3 donors over 24 hr following one-hour pulse treatment with ONX 0914.

Mean activity is reported with error bars representing the standard deviation from n = 3 replicates per donor.

https://doi.org/10.7554/eLife.27364.023
Figure 5—figure supplement 3
Western blots of LMP7 and β5 subunit levels during the ONX 0914 recovery time course in the SUP-B15 and MOLT-4 cell lines and in PBMCs from a healthy donor.
https://doi.org/10.7554/eLife.27364.024

Additional files

Supplementary File 1

Comparison of the reproducibility of the MSP-MS assay for technical triplicate samples.

Percent identity was compared across replicates for the intact library (‘no enzyme control’) and after the addition of the immunoproteasome as an example. The 8 hr time point was chosen to maximize the number of cleavages in the comparison.

https://doi.org/10.7554/eLife.27364.025
Supplementary File 2

Comparison of select ACC substrate Michaelis-Menten parameters.

https://doi.org/10.7554/eLife.27364.026
Supplementary File 3

Comparison of compound IC50 values in the MOLT-4 lysate obtained using the optimal fluorogenic substrates and the ProCISE assay.

https://doi.org/10.7554/eLife.27364.027
Transparent reporting form
https://doi.org/10.7554/eLife.27364.028

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