Kristofor Webb

Digestion and depletion of abundant proteins improves proteomic coverage.

Two major challenges in proteomics are the large number of proteins and their broad dynamic range in the cell. We exploited the abundance-dependent Michaelis-Menten kinetics of trypsin digestion to selectively digest and deplete abundant proteins with a method we call DigDeAPr. We validated the depletion mechanism with known yeast protein abundances, and we observed greater than threefold improvement in low-abundance human-protein identification and quantitation metrics. This methodology should be broadly applicable to many organisms, proteases and proteomic pipelines.Two major challenges in proteomics are the large number of proteins and their broad dynamic range within the cell. We exploited the abundance-dependent Michaelis-Menten kinetics of trypsin digestion to selectively digest and deplete abundant proteins with a method we call DigDeAPr. We validated the depletion mechanism with known yeast protein abundances and observed greater than 3-fold improvement in low abundance human protein identification and quantitation metrics. This methodology should be broadly applicable to many organisms, proteases, and proteomic pipelines.

The Unfolded Protein Response Triggers Site-Specific Regulatory Ubiquitylation of 40S Ribosomal Proteins.

Insults to ER homeostasis activate the unfolded protein response (UPR), which elevates protein folding and degradation capacity and attenuates protein synthesis. While a role for ubiquitin in regulating the degradation of misfolded ER-resident proteins is well described, ubiquitin-dependent regulation of translational reprogramming during the UPR remains uncharacterized. Using global quantitative ubiquitin proteomics, we identify evolutionarily conserved, site-specific regulatory ubiquitylation of 40S ribosomal proteins. We demonstrate that these events occur on assembled cytoplasmic ribosomes and are stimulated by both UPR activation and translation inhibition. We further show that ER stress-stimulated regulatory 40S ribosomal ubiquitylation occurs on a timescale similar to eIF2α phosphorylation, is dependent upon PERK signaling, and is required for optimal cell survival during chronic UPR activation. In total, these results reveal regulatory 40S ribosomal ubiquitylation as an important facet of eukaryotic translational control.

Structural basis for translational surveillance by the large ribosomal subunit-associated protein quality control complex

Significance All organisms have systems in place to ensure that aberrant nascent polypeptide chains are promptly dealt with before being released from ribosomes and posing harm to the cell. The ribosome-associated quality control complex (RQC), composed of the Ltn1 E3 ubiquitin ligase catalytic subunit and cofactors, has become a paradigm for understanding quality control in eukaryotes. However, exactly how RQC functions has remained unknown. Here, we determine the structure of the 60S subunit-bound RQC complex. The data provide critical insights into how RQC is able to selectively target aberrant nascent chains, while ignoring nascent chains in normally translating ribosomes. Furthermore, the structure shows the architecture of a ribosome-bound E3 ligase poised to mark nascent chains for degradation. All organisms have evolved mechanisms to manage the stalling of ribosomes upon translation of aberrant mRNA. In eukaryotes, the large ribosomal subunit-associated quality control complex (RQC), composed of the listerin/Ltn1 E3 ubiquitin ligase and cofactors, mediates the ubiquitylation and extraction of ribosome-stalled nascent polypeptide chains for proteasomal degradation. How RQC recognizes stalled ribosomes and performs its functions has not been understood. Using single-particle cryoelectron microscopy, we have determined the structure of the RQC complex bound to stalled 60S ribosomal subunits. The structure establishes how Ltn1 associates with the large ribosomal subunit and properly positions its E3-catalytic RING domain to mediate nascent chain ubiquitylation. The structure also reveals that a distinguishing feature of stalled 60S particles is an exposed, nascent chain-conjugated tRNA, and that the Tae2 subunit of RQC, which facilitates Ltn1 binding, is responsible for selective recognition of stalled 60S subunits. RQC components are engaged in interactions across a large span of the 60S subunit surface, connecting the tRNA in the peptidyl transferase center to the distally located nascent chain tunnel exit. This work provides insights into a mechanism linking translation and protein degradation that targets defective proteins immediately after synthesis, while ignoring nascent chains in normally translating ribosomes.

Recruitment of the 4EHP-GYF2 cap-binding complex to tetraproline motifs of tristetraprolin promotes repression and degradation of mRNAs with AU-rich elements

The zinc finger protein tristetraprolin (TTP) promotes translation repression and degradation of mRNAs containing AU-rich elements (AREs). Although much attention has been directed toward understanding the decay process and machinery involved, the translation repression role of TTP has remained poorly understood. Here we identify the cap-binding translation repression 4EHP-GYF2 complex as a cofactor of TTP. Immunoprecipitation and in vitro pull-down assays demonstrate that TTP associates with the 4EHP-GYF2 complex via direct interaction with GYF2, and mutational analyses show that this interaction occurs via conserved tetraproline motifs of TTP. Mutant TTP with diminished 4EHP-GYF2 binding is impaired in its ability to repress a luciferase reporter ARE-mRNA. 4EHP knockout mouse embryonic fibroblasts (MEFs) display increased induction and slower turnover of TTP-target mRNAs as compared to wild-type MEFs. Our work highlights the function of the conserved tetraproline motifs of TTP and identifies 4EHP-GYF2 as a cofactor in translational repression and mRNA decay by TTP.

Using the ubiquitin-modified proteome to monitor distinct and spatially restricted protein homeostasis dysfunction

Protein homeostasis dysfunction has been implicated in the development and progression of aging related human pathologies. There is a need for the establishment of quantitative methods to evaluate global protein homoeostasis function. As the ubiquitin (ub) proteasome system plays a key role in regulating protein homeostasis, we applied quantitative proteomic methods to evaluate the sensitivity of site-specific ubiquitylation events as markers for protein homeostasis dysfunction. Here, we demonstrate that the ub-modified proteome can exceed the sensitivity of engineered fluorescent reporters as a marker for proteasome dysfunction and can provide unique signatures for distinct proteome challenges which is not possible with engineered reporters. We demonstrate that combining ub-proteomics with subcellular fractionation can effectively separate degradative and regulatory ubiquitylation events on distinct protein populations. Using a recently developed potent inhibitor of the critical protein homeostasis factor p97/VCP, we demonstrate that distinct insults to protein homeostasis function can elicit robust and largely unique alterations to the ub-modified proteome. Taken together, we demonstrate that proteomic approaches to monitor the ub-modified proteome can be used to evaluate global protein homeostasis and can be used to monitor distinct functional outcomes for spatially separated protein populations.

Multimodal Mechanism of Action for the Cdc34 Acidic Loop A CASE STUDY FOR WHY UBIQUITIN-CONJUGATING ENZYMES HAVE LOOPS AND TAILS

Background: Ubiquitin-mediated proteolysis is the principal mechanism for regulating protein half-lives in cells. Results: Cdc34 promotes ubiquitin chain assembly onto protein substrates and contains an essential acidic loop near the active site. Conclusion: The Cdc34 acidic loop promotes both protein-protein interactions and catalysis of ubiquitin chain formation. Significance: These results uncover specific biochemical activities for the Cdc34 acidic loop. Together with ubiquitin ligases (E3), ubiquitin-conjugating enzymes (E2) are charged with the essential task of synthesizing ubiquitin chains onto protein substrates. Some 75% of the known E2s in the human proteome contain unique insertions in their primary sequences, yet it is largely unclear what effect these insertions impart on the ubiquitination reaction. Cdc34 is an important E2 with prominent roles in cell cycle regulation and signal transduction. The amino acid sequence of Cdc34 contains an insertion distal to the active site that is absent in most other E2s, yet this acidic loop (named for its four invariably conserved acidic residues) is critical for Cdc34 function both in vitro and in vivo. Here we have investigated how the acidic loop in human Cdc34 promotes ubiquitination, identifying two key molecular events during which the acidic loop exerts its influence. First, the acidic loop promotes the interaction between Cdc34 and its ubiquitin ligase partner, SCF. Second, two glutamic acid residues located on the distal side of the loop collaborate with an invariably conserved histidine on the proximal side of the loop to suppress the pKa of an ionizing species on ubiquitin or Cdc34 which greatly contributes to Cdc34 catalysis. These results demonstrate that insertions can guide E2s to their physiologically relevant ubiquitin ligases as well as provide essential modalities that promote catalysis.

Addendum: Digestion and depletion of abundant proteins improves proteomic coverage

Recently we reported improved proteome coverage and quantitation metrics for low abundance proteins within whole proteomes by incorporation of a novel digestion and depletion strategy prior to a standard shotgun proteomic analysis. Our goal was to improve the proteomic metrics of low abundance proteins by reducing proteolytic background, namely reducing the highly sampled peptides derived from high abundance proteins. Our method was successful in reducing proteolytic background. Conceptually we rationalized these gains resulted from a selective digestion and removal of abundant proteins, as peptides. Since our report, the mechanism by which our gains were achieved has been challenged in a Correspondence by Ye et al. (Nat. Methods, 2014). In response, we have reanalyzed our data in a peptide-centric manner and propose a refined kinetic mechanism consistent with established competitive substrate kinetics. Through a simplified derivation beginning with a classical Michaelis-Menten competitive substrate model and further quantitative analysis of our data, we provide a refined depletion mechanism that more accurately describes the complex mixtures we previously analyzed. Our revised qualitative expression describing depletion of early-generated peptides from proximal fast tryptic cleavage sites with high specificity constants (V/K) (Supplementary Note 1) is illustrated by the following equation: χA,depleted=(χA)t=0e(−VK)Atc∑(χn)t=0e(−VK)ntc−(χA)t=0e(−VK)Atd∑(χn)t=0e(−VK)ntd (1) where χA,depleted is the mole fraction of substrate A after complete (tc) and depletion (td) digestion times expressed as mole fraction of total substrate cleavage sites. So expressed, tryptic sites have different specificity constants as well as abundances. Substrate cleavage results in the generation of two shorter polypeptides that can be subsequently cleaved into more substrates over time. The relative cleavage rates are governed by each site’s relative specificity constant. From this perspective, we redefine the mechanism for depletion and enrichment of the digestion and depletion of abundant proteins (DigDeAPr) method. Early-generated peptides, derived from fast substrate sites (i.e. high V/K) within ~ 100 amino acids of each other, are removed at the point of our 10K molecular weight cutoff (MWCO) spin-filter depletion step. Clearing of these early-generated peptides prior to further digestion, provides an enrichment of peptides resulting from slower tryptic sites in the subsequent complete digestion step. By use of equation 1 we illustrate the expected adjustment in peptide abundance resulting from limited digestion and depletion (Figure 1a) as driven by the relative cleavage site specificity constants (V/K). When peptide abundance is considered between control and DigDeAPr runs, the expected trend is observed (Figure 1b, Supplementary Figure 1, and Supplementary Note 2) consistent with theory. Notably, the use of 10-fold more starting material and depletion of early-generated peptides acts to equalize the measured abundance of all peptides. Since peptide abundances are used to estimate protein abundance with shotgun proteomics,1–4 the equalization of peptides acts to also equalize the measurable abundance of proteins, as we found empirically in our initial analysis. Figure 1 Theoretical and empirical comparison of DigDeAPr mechanism. (a) Schematic of dominant mechanism from digestion and depletion based on the cleavage site specificity constant (V/K) for a given protease. The natural abundance of peptides from a complete ... Our depletion runs provide a defined, limited digestion time point for consideration of the aforementioned kinetic efficiencies through analysis of early- and later-generated peptides and fast and slow tryptic cleavage sites (Supplementary Note 3). Early-generated peptides should be depleted and have lower abundances after DigDeAPr when compared to control runs, while later-generated peptides should be enriched and have higher abundances. Using label-free chromatographic peak area ratios of peptides in both control and DigDeAPr runs, we quantified 13,628 and 13,112 peptides in HEK (Figure 2a) and yeast cells, respectively, that were used to classify peptides as early- or later-generated by their relative ratios. Both distributions show defined populations of peptides that were depleted (log2 ratio ≤ −1), unchanged (–1 < log2 ratio < 1), and enriched (log2 ratio ≥ 1). Focusing on the HEK peptide distribution, motif analysis of cleaved (Figure 2b) and missed cleaved (Figure 2c) tryptic sites on depleted peptides validates that early-generated peptides from proximal fast tryptic cleavage sites (< ~100 amino acids apart) are selectively removed during the 10 kDa depletion step (Supplementary Note 4). Similarly, tryptic motifs of enriched, later-generated peptides represent slow cleavage sites (Figure 2d) that remain uncleaved within polypeptides of greater than 10 kDa at the depletion time point. Thus, consideration of tryptic sites and peptides in the digestion and depletion mechanism is essential and illustrates the depletion and enrichment of peptides from fast and slow tryptic cleavage sites, respectively. Figure 2 Motif analysis to support DigDeAPr mechanism. (a) Distribution of quantified HEK peptide ratios using label-free peak area measurements from Fonslow et al. Tryptic site motif analysis using IceLogo6 and peptide ratios from analysis of HEK cells categorized ... With these peptide-centric considerations of abundance, when early- and later-generated peptides are now considered in our protein abundance analyses we notably still observe an abundance-based depletion and enrichment trend in both yeast and HEK cells: more abundant proteins have more early-generated peptides identified and less abundant proteins have more later-generated peptides identified (Supplementary Figure 5 and Note 5). Based on this data and our understanding of peptide sampling in shotgun proteomics,1 we conclude that our gains originate from analysis of a different population of enriched, later-generated peptides. That is, depletion of early-generated peptides from high abundance proteins removes enough proteolytic background to unmask and identify more later-generated peptides from low abundance proteins. Although we may not have explicitly depleted abundant proteins through digestion, in our reanalysis we find that depletion or enrichment of single peptides account for ~ 30% (1/slope = 0.298) of the observed protein abundance depletion or enrichment, respectively, explained by ~ 60% (R2 = 0.57) of the protein abundance measurements (Supplementary Figure 6 and Note 6). Additionally, we found a notable overlap in depleted, early-generated yeast peptides and “proteotypic” yeast peptides (Supplementary Figure 7 and Note 5). While “proteotypic” peptides can be used to robustly identify and quantify many proteins, they can also act as proteolytic background for other less abundant or less sampled proteins and peptides.5 These results collectively indicate that depletion of highly-sampled, abundant, easily-identified, “proteotypic” peptides has a similar effect as depleting abundant proteins to improve identification and quantification of peptides from low abundance proteins. With our refined view of peptide abundance changes and their correlation to protein changes, we propose a dominant mechanism by which our proteome coverage and quantitation gains are realized through digestion and depletion: depletion of early-generated peptides and enrichment of later-generated peptides equalizes measurable peptide abundances and unmasks less “proteotypic” peptides for improvements in low abundance protein identification and quantification. Based on these new contributing mechanisms, DigDeAPr instead represents digestion and depletion of abundantly sampled peptides and proteins through enrichment of less easily digested and identifiable proteins and peptides. Nonetheless, the combination of ten-fold more starting material with limited digestion and depletion remains a robust and promising method to remove the most easily and repeatedly detected peptides, clearing chromatographic, electrospray ionization, and mass spectrometer space for improvements in identification coverage and quantification of low abundance proteins. These new mechanistic insights suggest that varying limited digestion times in combination with the use of other proteases with different site specificity constants (V/K) and different MWCO filter sizes may hold the most potential to further improve coverage and quantitation of whole proteomes. We are excited about the future possibilities of similar methods and mechanistic investigations to further improve proteomic coverage and quantitation in shotgun proteomics.

Eavesdropping on PTM cross-talk through serial enrichment

Two approaches to serially enrich protein post-translational modifications allow the detection of multiple modifications in a single biological sample using mass spectrometry.

Decoys Untangle Complicated Redundancy and Reveal Targets of Circadian Clock F-Box Proteins

Decoy circadian clock proteins, which bind interacting proteins but prevent their degradation, reveal new functions and interactions of redundant E3 ubiquitin ligases. Eukaryotic circadian clocks utilize the ubiquitin proteasome system to precisely degrade clock proteins. In plants, the F-box-type E3 ubiquitin ligases ZEITLUPE (ZTL), FLAVIN-BINDING, KELCH REPEAT, F-BOX1 (FKF1), and LOV KELCH PROTEIN2 (LKP2) regulate clock period and couple the clock to photoperiodic flowering in response to end-of-day light conditions. To better understand their functions, we expressed decoy ZTL, FKF1, and LKP2 proteins that associate with target proteins but are unable to ubiquitylate their targets in Arabidopsis (Arabidopsis thaliana). These dominant-negative forms of the proteins inhibit the ubiquitylation of target proteins and allow for the study of ubiquitylation-independent and -dependent functions of ZTL, FKF1, and LKP2. We demonstrate the effects of expressing ZTL, FKF1, and LKP2 decoys on the circadian clock and flowering time. Furthermore, the decoy E3 ligases trap substrate interactions, and using immunoprecipitation-mass spectrometry, we identify interacting partners. We focus studies on the clock transcription factor CCA1 HIKING EXPEDITION (CHE) and show that ZTL interacts directly with CHE and can mediate CHE ubiquitylation. We also demonstrate that CHE protein is degraded in the dark and that degradation is reduced in a ztl mutant plant, showing that CHE is a bona fide ZTL target protein. This work increases our understanding of the genetic and biochemical roles for ZTL, FKF1, and LKP2 and also demonstrates an effective methodology for studying complicated genetic redundancy among E3 ubiquitin ligases.

Decoys reveal the genetic and biochemical roles of redundant plant E3 ubiquitin ligases

The ubiquitin proteasome system (UPS) is the main cellular route for protein degradation in plants and is important for a wide range of biological processes including daily and seasonal timing. The UPS relies on the action of E3 ubiquitin ligases to specifically recognize substrate proteins and facilitate their ubiquitylation. In plants, there are three major challenges that inhibit studies of E3 ligase function: 1) rampant genetic redundancy, 2) labile interactions between an E3 ligase and its cognate substrates, and 3) a lack of tools for rapid validation of bona fide substrates. To overcome these 3 challenges, we have developed a decoy method that allows for rapid genetic analysis of E3 ligases, in vivo identification of substrates using immunoprecipitation followed by mass spectrometry, and reconstitution of the ubiquitylation reaction in mammalian cells to rapidly validate potential substrates. We employ the strategy to study the plant F-box proteins, ZTL, LKP2, and FKF1 revealing differential genetic impacts on circadian clock period and seasonal flowering. We identify a group of circadian clock transcriptional regulators that interact with ZTL, LKP2, and FKF1 in vivo providing a host of potential substrates that have not been seen previously. We then validate one substrate of ZTL, the plant circadian clock transcription factor CHE, and show that ZTL mediates CHE ubiquitylation and that the levels of the CHE protein cycle in daily timecourses. This work further untangles the complicated genetic roles of this family of E3 ligases and suggests that ZTL is a master regulator of a diverse set of critical clock transcription factors. Furthermore, the method that is validated here can be tool employed widely to overcome traditional challenges in studying redundant plant E3 ubiquitin ligases.