Global Protein Turnover Research Articles

Global protein turnover quantification in Escherichia coli reveals cytoplasmic recycling under nitrogen limitation

Protein turnover is critical for proteostasis, but turnover quantification is challenging, and even in well-studied E. coli, proteome-wide measurements remain scarce. Here, we quantify the turnover rates of ~3200 E. coli proteins under 13 conditions by combining heavy isotope labeling with complement reporter ion quantification and find that cytoplasmic proteins are recycled when nitrogen is limited. We use knockout experiments to assign substrates to the known cytoplasmic ATP-dependent proteases. Surprisingly, none of these proteases are responsible for the observed cytoplasmic protein degradation in nitrogen limitation, suggesting that a major proteolysis pathway in E. coli remains to be discovered. Lastly, we show that protein degradation rates are generally independent of cell division rates. Thus, we present broadly applicable technology for protein turnover measurements and provide a rich resource for protein half-lives and protease substrates in E. coli, complementary to genomics data, that will allow researchers to study the control of proteostasis.

OsHYPK-mediated protein N-terminal acetylation coordinates plant development and abiotic stress responses in rice

N-terminal acetylation is one of the most common protein modifications in eukaryotes, and approximately 40% of human and plant proteomes are acetylated by ribosome-associated N-terminal acetyltransferase A (NatA) in a co-translational manner. However, the in vivo regulatory mechanism of NatA and the global impact of NatA-mediated N-terminal acetylation on protein fate remain unclear. Here, we identify Huntingtin Yeast partner K (HYPK), an evolutionarily conserved chaperone-like protein, as a positive regulator of NatA activity in rice. We found that loss of OsHYPK function leads to developmental defects in rice plant architecture but increased resistance to abiotic stresses, attributable to perturbation of the N-terminal acetylome and accelerated global protein turnover. Furthermore, we demonstrated that OsHYPK is also a substrate of NatA and that N-terminal acetylation of OsHYPK promotes its own degradation, probably through the Ac/N-degron pathway, which could be induced by abiotic stresses. Taken together, our findings suggest that the OsHYPK-NatA complex plays a critical role in coordinating plant development and stress responses by dynamically regulating NatA-mediated N-terminal acetylation and global protein turnover, which are essential for maintaining adaptive phenotypic plasticity in rice.

Cotranslational N-degron masking by acetylation promotes proteome stability in plants

N-terminal protein acetylation (NTA) is a prevalent protein modification essential for viability in animals and plants. The dominant executor of NTA is the ribosome tethered Nα-acetyltransferase A (NatA) complex. However, the impact of NatA on protein fate is still enigmatic. Here, we demonstrate that depletion of NatA activity leads to a 4-fold increase in global protein turnover via the ubiquitin-proteasome system in Arabidopsis. Surprisingly, a concomitant increase in translation, actioned via enhanced Target-of-Rapamycin activity, is also observed, implying that defective NTA triggers feedback mechanisms to maintain steady-state protein abundance. Quantitative analysis of the proteome, the translatome, and the ubiquitome reveals that NatA substrates account for the bulk of this enhanced turnover. A targeted analysis of NatA substrate stability uncovers that NTA absence triggers protein destabilization via a previously undescribed and widely conserved nonAc/N-degron in plants. Hence, the imprinting of the proteome with acetylation marks is essential for coordinating proteome stability.

ITARGEX analysis of yeast deletome reveals novel regulators of transcriptional buffering in S phase and protein turnover

Integrating omics data with quantification of biological traits provides unparalleled opportunities for discovery of genetic regulators by in silico inference. However, current approaches to analyze genetic-perturbation screens are limited by their reliance on annotation libraries for prioritization of hits and subsequent targeted experimentation. Here, we present iTARGEX (identification of Trait-Associated Regulatory Genes via mixture regression using EXpectation maximization), an association framework with no requirement of a priori knowledge of gene function. After creating this tool, we used it to test associations between gene expression profiles and two biological traits in single-gene deletion budding yeast mutants, including transcription homeostasis during S phase and global protein turnover. For each trait, we discovered novel regulators without prior functional annotations. The functional effects of the novel candidates were then validated experimentally, providing solid evidence for their roles in the respective traits. Hence, we conclude that iTARGEX can reliably identify novel factors involved in given biological traits. As such, it is capable of converting genome-wide observations into causal gene function predictions. Further application of iTARGEX in other contexts is expected to facilitate the discovery of new regulators and provide observations for novel mechanistic hypotheses regarding different biological traits and phenotypes.

Proteasome-Bound UCH37/UCHL5 Debranches Ubiquitin Chains to Promote Degradation

The linkage, length, and architecture of ubiquitin (Ub) chains are all important variables in providing tight control over many biological paradigms. There are clear roles for branched architectures in regulating proteasome-mediated degradation, but the proteins that selectively recognize and process these atypical chains are unknown. Here, using synthetic and enzyme-derived ubiquitin chains along with intact mass spectrometry, we report that UCH37/UCHL5, a proteasome-associated deubiquitinase, cleaves K48 branched chains. The activity and selectivity toward branched chains is markedly enhanced by the proteasomal Ub receptor RPN13/ADRM1. Using reconstituted proteasome complexes, we find that chain debranching promotes degradation of substrates modified with branched chains under multi-turnover conditions. These results are further supported by proteome-wide pulse-chase experiments, which show that the loss of UCH37 activity impairs global protein turnover. Our work therefore defines UCH37 as a debranching deubiquitinase important for promoting proteasomal degradation.

Long-lived metabolic enzymes in the crystalline lens identified by pulse-labeling of mice and mass spectrometry.

The lenticular fiber cells are comprised of extremely long-lived proteins while still maintaining an active biochemical state. Dysregulation of these activities has been implicated in diseases such as age-related cataracts. However, the lenticular protein dynamics underlying health and disease is unclear. We sought to measure the global protein turnover rates in the eye using nitrogen-15 labeling of mice and mass spectrometry. We measured the 14N/15N-peptide ratios of 248 lens proteins, including Crystallin, Aquaporin, Collagen and enzymes that catalyze glycolysis and oxidation/reduction reactions. Direct comparison of lens cortex versus nucleus revealed little or no 15N-protein contents in most nuclear proteins, while there were a broad range of 14N/15N ratios in cortex proteins. Unexpectedly, like Crystallins, many enzymes with relatively high abundance in nucleus were also exceedingly long-lived. The slow replacement of these enzymes in spite of young age of mice suggests their potential roles in age-related metabolic changes in the lens.

Cross-species Comparison of Proteome Turnover Kinetics

The constitutive process of protein turnover plays a key role in maintaining cellular homeostasis. Recent technological advances in mass spectrometry have enabled the measurement of protein turnover kinetics across the proteome. However, it is not known if turnover kinetics of individual proteins are highly conserved or if they have evolved to meet the physiological demands of individual species. Here, we conducted systematic analyses of proteome turnover kinetics in primary dermal fibroblasts isolated from eight different rodent species. Our results highlighted two trends in the variability of proteome turnover kinetics across species. First, we observed a decrease in cross-species correlation of protein degradation rates as a function of evolutionary distance. Second, we observed a negative correlation between global protein turnover rates and maximum lifespan of the species. We propose that by reducing the energetic demands of continuous protein turnover, long-lived species may have evolved to lessen the generation of reactive oxygen species and the corresponding oxidative damage over their extended lifespans.

SENP3 regulates the global protein turnover and the Sp1 level via antagonizing SUMO2/3-targeted ubiquitination and degradation.

SUMOylation is recently found to function as a targeting signal for the degradation of substrates through the ubiquitin-proteasome system. RNF4 is the most studied human SUMO-targeted ubiquitin E3 ligase. However, the relationship between SUMO proteases, SENPs, and RNF4 remains obscure. There are limited examples of the SENP regulation of SUMO2/3-targeted proteolysis mediated by RNF4. The present study investigated the role of SENP3 in the global protein turnover related to SUMO2/3-targeted ubiquitination and focused in particular on the SENP3 regulation of the stability of Sp1. Our data demonstrated that SENP3 impaired the global ubiquitination profile and promoted the accumulation of many proteins. Sp1, a cancer-associated transcription factor, was among these proteins. SENP3 increased the level of Sp1 protein via antagonizing the SUMO2/3-targeted ubiquitination and the consequent proteasome-dependent degradation that was mediated by RNF4. De-conjugation of SUMO2/3 by SENP3 attenuated the interaction of Sp1 with RNF4. In gastric cancer cell lines and specimens derived from patients and nude mice, the level of Sp1 was generally increased in parallel to the level of SENP3. These results provided a new explanation for the enrichment of the Sp1 protein in various cancers, and revealed a regulation of SUMO2/3 conjugated proteins whose levels may be tightly controlled by SENP3 and RNF4.

C&EN Webinars - Comparative Study of Global Protein Turnover in Tissues and Cell Lines

C&EN Webinars - Comparative Study of Global Protein Turnover in Tissues and Cell Lines

Dysfunction of protein homeostasis in myotonic dystrophies.

Neuromuscular diseases Myotonic Dystrophies type 1 and type 2 (DM1 and DM2) are caused by unstable CTG and CCTG repeat expansions and have highly complex molecular mechanisms. DM1 is caused by the expansion of CTG repeats in the 3' UTR of the gene coding for Dystrophia Myotonica-Protein Kinase (DMPK). In DM2, intronic CCTG repeats are located in a gene encoding the Zinc Finger Protein 9 (ZNF9, also known as Cellular Nucleic Acid Binding Protein, CNBP). Both expansions cause pathologies through RNA CUG and CCUG repeats, which have toxic effects on the processing of many RNAs in the patients' tissues. The pathogenic role of CUG and CCUG repeats in the mis-regulation of alternative splicing, mediated by RNA-binding proteins CUGBP1 and MBNL1, has been discussed in a number of excellent reviews. Recent reports suggest that mutant RNA repeats affect several other RNA-binding proteins such as Staufen1 and the DEAD-box RNA helicase p68 (DDX5). Since CUGBP1, Staufen1 and p68 have many functions in cytoplasm, including regulation of protein translation, it is predicted that the alterations of these proteins in DM cells might have a toxic effect on global protein turnover. In this mini-review, we will summarize observations showing the role of RNA-binding proteins, CUGBP1 and ZNF9, in protein turnover in DM1 and in DM2. We will also discuss a possible role of misbalanced protein turnover in the age-dependent progression of DM1 and in a late onset of DM2.

Destabilization of Heterologous Proteins Mediated by the GSK3β Phosphorylation Domain of the β-Catenin Protein

Background and Aims: Wnt/β-catenin signaling plays important roles in development and cellular processes. The hallmark of canonical Wnt signaling activation is the stabilization of β-catenin protein in cytoplasm and/or nucleus. The stability of β-catenin is the key to its biological functions and is controlled by the phosphorylation of its amino-terminal degradation domain. Aberrant activation of β-catenin signaling has been implicated in the development of human cancers. It has been recently suggested that GSK3βmay play an essential role in regulating global protein turnover. Here, we investigate if the GSK3β phosphorylation site-containing degradation domain of β-catenin is sufficient to destabilize heterologous proteins. Methods and Results: We engineer chimeric proteins by fusing β-catenin degradation domain at the N- and/or C-termini of the enhanced green fluorescent protein (eGFP). In both transient and stable expression experiments, the chimeric GFP proteins exhibit a significantly decreased stability, which can be effectively antagonized by lithium and Wnt1. An activating mutation in the destruction domain significantly stabilizes the fusion protein. Furthermore, GSK3 inhibitor SB-216763 effectively increases the GFP signal of the fusion protein. Conversely, the inhibition of Wnt signaling with tankyrase inhibitor XAV939 results in a decrease in GFP signal of the fusion proteins, while these small molecules have no significant effects on the mutant destruction domain-GFP fusion protein. Conclusion: Our findings strongly suggest that the β-catenin degradation domain may be sufficient to destabilize heterologous proteins in Wnt signaling-dependent manner. It is conceivable that the chimeric GFP proteins may be used as a functional reporter to measure the dynamic status of β-catenin signaling, and to identify potential anticancer drugs that target β-catenin signaling.

Abstract C82: Identification and preclinical characterization of inhibitors of the ubiquitin-activating enzymes UBA1 and UBA6.

Abstract Millennium Pharmaceuticals, Inc. is dedicated to the discovery and development of novel oncology therapeutics in the area of protein homeostasis. Here we report the identification and characterization of compounds that target the ubiquitin activating enzymes, UBA1 and UBA6. These compounds are mechanism based inhibitors that inactivate the ubiquitin E1 enzymes by forming a ubiquitin compound adduct that remains tightly associated with the E1 adenylate binding site. Treatment of cells with these inhibitors results in cellular effects consistent with known Uba1 biology including rapid loss of E2 ubiquitin thioesters, loss of total ubiquitin conjugates, and accumulation of many ubiquitin proteasome system substrates. Following prolonged treatment, cells primarily arrest in the G2 phase of the cell cycle and ultimately undergo apoptosis. Reflecting the extensive cellular roles of ubiquitin, the compounds also impact global protein turnover, ER stress and DNA damage repair. UBA1 inhibition impairs ubiquitination of PCNA and the Fanconia Anemia protein FANCD2 leading to defective repair of UV induced DNA damage. UBA1 inhibition impacts numerous biological pathways relevant to cancer, results in apoptosis in vitro and is capable of inhibiting tumor growth in mouse xenografts in vivo. These data implicate UBA1 as a target for the treatment of cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C82.

Measurement of human plasma proteome dynamics with 2H 2O and liquid chromatography tandem mass spectrometry

Dysfunction of protein turnover is a feature of many human diseases, and proteins are substrates in important biological processes. Currently, no method exists for the measurement of global protein turnover (i.e., proteome dynamics) that can be applied in humans. Here we describe the use of metabolic labeling with deuterium ( 2H) from 2H 2O and liquid chromatography tandem mass spectrometry (LC–MS/MS) analysis of mass isotopomer patterns to measure protein turnover. We show that the positions available for 2H label incorporation in vivo can be calculated using peptide sequence. The isotopic incorporation values calculated by combinatorial analysis of mass isotopomer patterns in peptides correlate very closely with values established for individual amino acids. Inpatient and outpatient heavy water labeling protocols resulted in 2H label incorporation sufficient for reproducible quantitation in humans. Replacement rates were similar for peptides deriving from the same protein. Using a kinetic model to account for the time course of each individual’s 2H 2O enrichment curves, dynamics of approximately 100 proteins with half-lives ranging from 0.4 to 40 days were measured using 8 μl of plasma. The measured rates were consistent with literature values. This method can be used to measure in vivo proteome homeostasis in humans in disease and during therapeutic interventions.

Comparative Proteomic Analysis of Methanothermobacter themautotrophicus ΔH in Pure Culture and in Co-Culture with a Butyrate-Oxidizing Bacterium

To understand the physiological basis of methanogenic archaea living on interspecies H2 transfer, the protein expression of a hydrogenotrophic methanogen, Methanothermobacter thermautotrophicus strain ΔH, was investigated in both pure culture and syntrophic coculture with an anaerobic butyrate oxidizer Syntrophothermus lipocalidus strain TGB-C1 as an H2 supplier. Comparative proteomic analysis showed that global protein expression of methanogen cells in the model coculture was substantially different from that of pure cultured cells. In brief, in syntrophic coculture, although methanogenesis-driven energy generation appeared to be maintained by shifting the pathway to the alternative methyl coenzyme M reductase isozyme I and cofactor F420-dependent process, the machinery proteins involved in carbon fixation, amino acid synthesis, and RNA/DNA metabolisms tended to be down-regulated, indicating restrained cell growth rather than vigorous proliferation. In addition, our proteome analysis revealed that α subunits of proteasome were differentially acetylated between the two culture conditions. Since the relevant modification has been suspected to regulate proteolytic activity of the proteasome, the global protein turnover rate could be controlled under syntrophic growth conditions. To our knowledge, the present study is the first report on N-acetylation of proteasome subunits in methanogenic archaea. These results clearly indicated that physiological adaptation of hydrogenotrophic methanogens to syntrophic growth is more complicated than that of hitherto proposed.

Healthy ageing through regulated proteostasis

During metazoan life, protein homeostasis (proteostasis) declines with age due to impaired proteasome activity. The signalling pathways activated by growth factors that regulate proteostasis and their importance to ageing are just emerging. In this issue, Liu et al report a crucial role of epidermal growth factor (EGF) in regulating Caenorhabditis elegans lifespan through the RAF/MAPK pathway. EGF signalling activates the ubiquitin–proteosome system (UPS) and represses the chaperone machinery by modulating the expression of several ageing‐related genes (gerontogenes). This strategic switch in controlling global protein turnover provides novel insights into the molecular mechanisms driving ageing in multi‐cellular organisms.

Determination of Global Protein Turnover in Stressed Mycobacterium Cells Using Hybrid-Linear Ion Trap-Fourier Transform Mass Spectrometry

We determined the global protein turnover profiles for Mycobacterium smegmatis under acid shock and iron starvation conditions using a simple (15)N isotope doping technique and a complete medium replacement method for chasing. We used a high-resolution hybrid-linear ion trap-Fourier transform mass spectrometer coupled with nanoliquid chromatography separation to measure protein turnover values for 151 proteins over a dynamic range of 3 orders of magnitude ranging from about 0.2 to 500. Of these 151 proteins, 31 had significant protein turnover changes (p <0.05) at both stress conditions and had protein turnover values increased or decreased by more than 2-fold under at least one stress condition. Protein turnover increased under acid shock for 28 of the 31 proteins but decreased under iron starvation for all the 31 proteins. Only two proteins had protein turnover lowered by more than 2-fold (p <0.05) under both stress conditions, including an ATP synthase F1 beta subunit (MSMEG4921; AtpD) and a catalase/peroxidase (MSMEG6346; KatG). KatG is required for in vivo activation of isoniazid to be bacterialcidal. Decrease of KatG protein turnover under both stress conditions supports the view that isoniazid may induce a dormancy program in mycobacteria, which in turn limits the efficacy of this drug against dormant subpopulation of mycobacteria. Thus, measuring protein turnover in stressed Mycobacterium cells has implications in understanding drug action and resistance mechanisms.

Global Organization and Function of Mammalian Cytosolic Proteasome Pools: Implications for PA28 and 19S Regulatory Complexes

Proteolytic activity of the 20S proteasome is regulated by activators that govern substrate movement into and out of the catalytic chamber. However, the physiological relationship between activators, and hence the relative role of different proteasome species, remains poorly understood. To address this problem, we characterized the total pool of cytosolic proteasomes in intact and functional form using a single-step method that bypasses the need for antibodies, proteasome modification, or column purification. Two-dimensional Blue Native(BN)/SDS-PAGE and tandem mass spectrometry simultaneously identified six native proteasome populations in untreated cytosol: 20S, singly and doubly PA28-capped, singly 19S-capped, hybrid, and doubly 19S-capped proteasomes. All proteasome species were highly dynamic as evidenced by recruitment and exchange of regulatory caps. In particular, proteasome inhibition with MG132 markedly stimulated PA28 binding to exposed 20S alpha-subunits and generated doubly PA28-capped and hybrid proteasomes. PA28 recruitment virtually eliminated free 20S particles and was blocked by ATP depletion. Moreover, inhibited proteasomes remained stably associated with distinct cohorts of partially degraded fragments derived from cytosolic and ER substrates. These data establish a versatile platform for analyzing substrate-specific proteasome function and indicate that PA28 and 19S activators cooperatively regulate global protein turnover while functioning at different stages of the degradation cycle.