Global analysis of cellular protein translation by pulsed SILAC

Current methods for system‐wide gene expression analysis detect changes in mRNA abundance, but neglect regulation at the level of translation. Pulse labeling with stable isotopes has been used to measure protein turnover rates, but this does not directly provide information about translation rates. Here, we developed pulsed stable isotope labeling by amino acids in cell culture (pSILAC) with two heavy isotope labels to directly quantify protein translation on a proteome‐wide scale. We applied the method to cellular iron homeostasis as a model system and demonstrate that it can confidently identify proteins that are translationally regulated by iron availability.     

Harmonizing Labeling and Analytical Strategies to Obtain Protein Turnover Rates in Intact Adult Animals

Changes in the abundance of individual proteins in the proteome can be elicited by modulation of protein synthesis (the rate of input of newly synthesized proteins into the protein pool) or degradation (the rate of removal of protein molecules from the pool). A full understanding of proteome changes therefore requires a definition of the roles of these two processes in proteostasis, collectively known as protein turnover. Because protein turnover occurs even in the absence of overt changes in pool abundance, turnover measurements necessitate monitoring the flux of stable isotope–labeled precursors through the protein pool such as labeled amino acids or metabolic precursors such as ammonium chloride or heavy water. In cells in culture, the ability to manipulate precursor pools by rapid medium changes is simple, but for more complex systems such as intact animals, the approach becomes more convoluted. Individual methods bring specific complications, and the suitability of different methods has not been comprehensively explored. In this study, we compare the turnover rates of proteins across four mouse tissues, obtained from the same inbred mouse strain maintained under identical husbandry conditions, measured using either [¹³C₆]lysine or [²H₂]O as the labeling precursor. We show that for long-lived proteins, the two approaches yield essentially identical measures of the first-order rate constant for degradation. For short-lived proteins, there is a need to compensate for the slower equilibration of lysine through the precursor pools. We evaluate different approaches to provide that compensation. We conclude that both labels are suitable, but careful determination of precursor enrichment kinetics in amino acid labeling is critical and has a considerable influence on the numerical values of the derived protein turnover rates.     

Determining degradation and synthesis rates of arabidopsis proteins using the kinetics of progressive 15N labeling of two-dimensional gel-separated protein spots

The growth and development of plant tissues is associated with an ordered succession of cellular processes that are reflected in the appearance and disappearance of proteins. The control of the kinetics of protein turnover is central to how plants can rapidly and specifically alter protein abundance and thus molecular function in response to environmental or developmental cues. However, the processes of turnover are largely hidden during periods of apparent steady-state protein abundance, and even when proteins accumulate it is unclear whether enhanced synthesis or decreased degradation is responsible. We have used a (15)N labeling strategy with inorganic nitrogen sources coupled to a two-dimensional fluorescence difference gel electrophoresis and mass spectrometry analysis of two-dimensional IEF/SDS-PAGE gel spots to define the rate of protein synthesis (K(S)) and degradation (K(D)) of Arabidopsis cell culture proteins. Through analysis of MALDI-TOF/TOF mass spectra from 120 protein spots, we were able to quantify K(S) and K(D) for 84 proteins across six functional groups and observe over 65-fold variation in protein degradation rates. K(S) and K(D) correlate with functional roles of the proteins in the cell and the time in the cell culture cycle. This approach is based on progressive (15)N labeling that is innocuous for the plant cells and, because it can be used to target analysis of proteins through the use of specific gel spots, it has broad applicability.     

The sterol‐binding activity of PATHOGENESIS‐RELATED PROTEIN 1 reveals the mode of action of an antimicrobial protein

Pathogenesis‐related proteins played a pioneering role 50 years ago in the discovery of plant innate immunity as a set of proteins that accumulated upon pathogen challenge. The most abundant of these proteins, PATHOGENESIS‐RELATED 1 (PR‐1) encodes a small antimicrobial protein that has become, as a marker of plant immune signaling, one of the most referred to plant proteins. The biochemical activity and mode of action of PR‐1 proteins has remained elusive, however. Here, we provide genetic and biochemical evidence for the capacity of PR‐1 proteins to bind sterols, and demonstrate that the inhibitory effect on pathogen growth is caused by the sequestration of sterol from pathogens. In support of our findings, sterol‐auxotroph pathogens such as the oomycete Phytophthora are particularly sensitive to PR‐1, whereas sterol‐prototroph fungal pathogens become highly sensitive only when sterol biosynthesis is compromised. Our results are in line with previous findings showing that plants with enhanced PR‐1 expression are particularly well protected against oomycete pathogens.     

Accumulation of “Old Proteins” and the Critical Need for MS‐based Protein Turnover Measurements in Aging and Longevity

Aging and age‐related diseases are accompanied by proteome remodeling and progressive declines in cellular machinery required to maintain protein homeostasis (proteostasis), such as autophagy, ubiquitin‐mediated degradation, and protein synthesis. While many studies have focused on capturing changes in proteostasis, the identification of proteins that evade these cellular processes has recently emerged as an approach to studying the aging proteome. With advances in proteomic technology, it is possible to monitor protein half‐lives and protein turnover at the level of individual proteins in vivo. For large‐scale studies, these technologies typically include the use of stable isotope labeling coupled with MS and comprehensive assessment of protein turnover rates. Protein turnover studies have revealed groups of highly relevant long‐lived proteins (LLPs), such as the nuclear pore complexes, extracellular matrix proteins, and protein aggregates. Here, the role of LLPs during aging and age‐related diseases and the methods used to identify and quantify their changes are reviewed. The methods available to conduct studies of protein turnover, used in combination with traditional proteomic methods, will enable the field to perform studies in a systems biology context, as changes in proteostasis may not be revealed in studies that solely measure differential protein abundances.     

The use of heavy nitrogen in quantitative proteomics experiments in plants

In the growing field of plant systems biology, there is an undisputed need for methods allowing accurate quantitation of proteins and metabolites. As autotrophic organisms, plants can easily metabolize different nitrogen isotopes, resulting in proteins and metabolites with distinct molecular mass that can be separated on a mass spectrometer. In comparative quantitative experiments, treated and untreated samples are differentially labeled by nitrogen isotopes and jointly processed, thereby minimizing sample-to-sample variation. In recent years, heavy nitrogen labeling has become a widely used strategy in quantitative proteomics and novel approaches have been developed for metabolite identification. Here, we present an overview of currently used experimental strategies in heavy nitrogen labeling in plants and provide background on the history and function of this quantitation technique.     

Proteomic analysis of Arabidopsis halleri shoots in response to the heavy metals cadmium and zinc and rhizosphere microorganisms

Arabidopsis halleri has the rare ability to colonize heavy metal‐polluted sites and is an emerging model for research on adaptation and metal hyperaccumulation. The aim of this study was to analyze the effect of plant–microbe interaction on the accumulation of cadmium (Cd) and zinc (Zn) in shoots of an ecotype of A. halleri grown in heavy metal‐contaminated soil and to compare the shoot proteome of plants grown solely in the presence of Cd and Zn or in the presence of these two metals and the autochthonous soil rhizosphere‐derived microorganisms. The results of this analysis emphasized the role of plant–microbe interaction in shoot metal accumulation. Differences in protein expression pattern, identified by a proteomic approach involving 2‐DE and MS, indicated a general upregulation of photosynthesis‐related proteins in plants exposed to metals and to metals plus microorganisms, suggesting that metal accumulation in shoots is an energy‐demanding process. The analysis also showed that proteins involved in plant defense mechanisms were downregulated indicating that heavy metals accumulation in leaves supplies a protection system and highlights a cross‐talk between heavy metal signaling and defense signaling.     

The Cost of Maintenance Processes in Plant Cells

The most important maintenance processes in plants are proteinturnover and active transport processes to maintain certainion concentrations in the cells. In this paper an attempt ismade to calculate the total energy cost of these processes fromwhat is known about their specific costs and what has been observedabout their rates. Because of insufficient reliable data aboutrates of individual maintenance processes, only approximatevalues can be obtained. The average turnover rate of leaf proteins may be about 100mg protein per g proteins per day at normal temperature in leavesassimilating at moderate light intensities. This process consumes28–53 mg glucose per g protein per day, which equals 7–13mg glucose per g dry weight per day in leaves. It is likelythat the rates of protein turnover and of CO₂-assimilation arerelated. The cost of maintaining ion concentrations is estimatedto be about 6–10 mg glucose per g dry weight per day inleaves. The sum of these figures is lower than is indicatedby measurements of maintenance respiration. One reason for theunderestimation may be that the protein turnover rates usedin the calculations apply to plants with lower photosyntheticrates than the plants in which the maintenance respiration wasmeasured. Effects of water stress and salinity, temperatureand other environmental factors on the rate of maintenance processesare discussed. The consumption of assimilates for maintenance of plant cellsis a significant, negative factor in plant productivity. A betterunderstanding of the maintenance processes may give a clue howto manipulate plant characteristics or the environment to reducethe amount of assimilates consumed in these processes. It issuggested that reduction in protein turnover rates may be onesuch manipulation.     

Impact of oxidative stress on the function,abundance, and turnover of the Arabidopsis 80S cytosolic ribosome

Abiotic stress in plants causes accumulation of reactive oxygen species (ROS) leading to the need for new protein synthesis to defend against ROS and to replace existing proteins that are damaged by oxidation. Functional plant ribosomes are critical for these activities, however we know little about the impact of oxidative stress on plant ribosome abundance, turnover, and function. Using Arabidopsis cell culture as a model system, we induced oxidative stress using 1 µm of H₂O₂ or 5 µm menadione to more than halve cell growth rate and limit total protein content. We show that ribosome content on a total cell protein basis decreased in oxidatively stressed cells. However, overall protein synthesis rates on a ribosome abundance basis showed the resident ribosomes retained their function in oxidatively stressed cells. ¹⁵N progressive labelling was used to calculate the rate of ribosome synthesis and degradation to track the fate of 62 r‐proteins. The degradation rates and the synthesis rates of most r‐proteins slowed following oxidative stress leading to an ageing population of ribosomes in stressed cells. However, there were exceptions to this trend; r‐protein RPS14C doubled its degradation rate in both oxidative treatments. Overall, we show that ribosome abundance decreases and their age increases with oxidative stress in line with loss of cell growth rate and total cellular protein amount, but ribosome function of the ageing ribosomes appeared to be maintained concomittently with differences in the turnover rate and abundance of specific ribosomal proteins. Data are available via ProteomeXchange with identifier PXD012840.     

Activity profiling of vacuolar processing enzymes reveals a role for VPE during oomycete infection

Vacuolar processing enzymes (VPEs) are important cysteine proteases that are implicated in the maturation of seed storage proteins, and programmed cell death during plant–microbe interactions and development. Here, we introduce a specific, cell‐permeable, activity‐based probe for VPEs. This probe is highly specific for all four Arabidopsis VPEs, and labeling is activity‐dependent, as illustrated by sensitivity for inhibitors, pH and reducing agents. We show that the probe can be used for in vivo imaging and displays multiple active isoforms of VPEs in various tissues and in both monocot and dicot plant species. Thus, VPE activity profiling is a robust, simple and powerful tool for plant research for a wide range of applications. Using VPE activity profiling, we discovered that VPE activity is increased during infection with the oomycete pathogen Hyaloperonospora arabidopsidis (Hpa). The enhanced VPE activity is host‐derived and EDS1‐independent. Sporulation of Hpa is reduced on vpe mutant plants, demonstrating a role for VPE during compatible interactions that is presumably independent of programmed cell death. Our data indicate that, as an obligate biotroph, Hpa takes advantage of increased VPE activity in the host, e.g. to mediate protein turnover and nutrient release.     

Green fluorescent protein fusions to Arabidopsis fimbrin 1 for spatio-temporal imaging of F-actin dynamics in roots

The visualization of green fluorescent protein (GFP) fusions with microtubule or actin filament (F-actin) binding proteins has provided new insights into the function of the cytoskeleton during plant development. For studies on actin, GFP fusions to talin have been the most generally used reporters. Although GFP-Talin has allowed in vivo F-actin imaging in a variety of plant cells, its utility in monitoring F-actin in stably transformed plants is limited particularly in developing roots where interesting actin dependent cell processes are occurring. In this study, we created a variety of GFP fusions to Arabidopsis Fimbrin 1 (AtFim1) to explore their utility for in vivo F-actin imaging in root cells and to better understand the actin binding properties of AtFim1 in living plant cells. Translational fusions of GFP to full-length AtFim1 or to some truncated variants of AtFim1 showed filamentous labeling in transient expression assays. One truncated fimbrin-GFP fusion was capable of labeling distinct filaments in stably transformed Arabidopsis roots. The filaments decorated by this construct were highly dynamic in growing root hairs and elongating root cells and were sensitive to actin disrupting drugs. Therefore, the fimbrin-GFP reporters we describe in this study provide additional tools for studying the actin cytoskeleton during root cell development. Moreover, the localization of AtFim1-GFP offers insights into the regulation of actin organization in developing roots by this class of actin cross-linking proteins.     

棕榈酰化蛋白及蛋白质的棕榈酰化研究进展

蛋白质S-棕榈酰化是最常见的具有16碳脂肪酸棕榈酸酯的脂质修饰形式,调节蛋白质的运输和功能。文中主要概括从植物到哺乳动物中发现的具有棕榈酰基转移酶活性的保守DHHC蛋白家族,并介绍蛋白质棕榈酰化的研究方法,及检测棕榈酰化蛋白质的位点预测方法(CSS-Palm、NBA-Palm、TermiNator2)、放射性标记法(用3H棕榈酸酯或125I-IC16棕榈酸酯)和非放射性标记法(化学标记和质谱法),总结蛋白棕榈酰化的抑制技术以及抑制剂类型(包括2-溴棕榈酸酯、浅蓝菌素和衣霉素)。同时概括蛋白棕榈酰化在植物胁迫中的响应,展望其在植物抗逆中的应用前景。     

The divergence and positive selection of the plant‐specific BURP‐containing protein family

BURP domain‐containing proteins belong to a plant‐specific protein family and have diverse roles in plant development and stress responses. However, our understanding about the genetic divergence patterns and evolutionary rates of these proteins remain inadequate. In this study, 15 plant genomes were explored to elucidate the genetic origins, divergence, and functions of these proteins. One hundred and twenty‐five BURP protein‐encoding genes were identified from four main plant lineages, including 13 higher plant species. The absence of BURP family genes in unicellular and multicellular algae suggests that this family (1) appeared when plants shifted from relatively stable aquatic environments to land, where conditions are more variable and stressful, and (2) is critical in the adaptation of plants to adverse environments. Promoter analysis revealed that several responsive elements to plant hormones and external environment stresses are concentrated in the promoter region of BURP protein‐encoding genes. This finding confirms that these genes influence plant stress responses. Several segmentally and tandem‐duplicated gene pairs were identified from eight plant species. Thus, in general, BURP domain‐containing genes have been subject to strong positive selection, even though these genes have conformed to different expansion models in different species. Our study also detected certain critical amino acid sites that may have contributed to functional divergence among groups or subgroups. Unexpectedly, all of the critical amino acid residues of functional divergence and positive selection were exclusively located in the C‐terminal region of the BURP domain. In conclusion, our results contribute novel insights into the genetic divergence patterns and evolutionary rates of BURP proteins.     

Measurement of protein turnover rates by heavy water labeling of nonessential amino acids

In vivo measurements of protein synthesis using isotope-labeled amino acids (AAs) are hampered by the heterogeneity of AA pools and, for slow turnover proteins, the difficulty and expense of long-term labeling. Continuous oral heavy water (2H2O) labeling can safely maintain stable body water 2H enrichments for weeks or months. 2H is metabolically incorporated into C-H bonds of nonessential AAs (NEAAs) and hence into proteins. No posttranslational label exchange occurs, so 2H incorporation into protein NEAAs, in principle, reports on protein synthesis. Here, we show by mass isotopomer distribution analysis (MIDA) of 2H2O-labeled rodent tissue proteins that metabolic 2H flux into C-H bonds of Ala, Gly, or Gln used for protein synthesis is nearly complete. By 2H2O labeling of rodents, turnover of bone and muscle mixed proteins was quantified and stimulation of liver collagen synthesis by CCl4 was detected. Kinetics of several human serum proteins were also measured, reproducing published t1/2 estimates. Plateau enrichments in Ala varied among different proteins. Moderate amounts of protein, isolated chromatographically or electrophoretically, sufficed for kinetic analyses. In conclusion, 2H2O labeling permits sensitive, quantitative, operationally simple measurements of protein turnover in vivo by the rise-to-plateau approach, especially for proteins with slow constitutive turnover.     

Turnover of nitrogenase and leghemoglobin in root nodules of Pisum sativum

Turnover rates of the two nitrogenase components and leghemoglobin in root nodules of pea plants nodulated with Rhizobium leguminosarum were determined with three different methods: 1, Kinetics of 35S incorporation into protein; 2, pulse-chase experiments; 3, chloramphenicol inhibition of bacteroid protein synthesis. Methods 1 and 3 revealed that the turnover rates of the two nitrogenase components and leghemoglobin are identical to the average rate of bacteroid and plant nodule protein turnover. The t1/2 times of component I and II and leghemoglobin were about 2 days. Pulse-chase experiments with 35SO(2-)4 appeared to be rather unsuitable for determination of turnover rates in pea root nodules.     

Efficient ASK‐assisted system for expression and purification of plant F‐box proteins

Ubiquitin‐mediated protein degradation plays an essential role in plant growth and development as well as responses to environmental and endogenous signals. F‐box protein is one of the key components of the SCF (SKP1‐CUL1‐F‐box protein) E3 ubiquitin ligase complex, which recruit specific substrate proteins for subsequent ubiquitination and 26S proteasome‐mediated degradation to regulate developmental processes and signaling networks. However, it is not easy to obtain purified F‐box proteins with high activity due to their unstable protein structures. Here, we found that Arabidopsis SKP‐like proteins (ASKs) can significantly improve soluble expression of F‐box proteins and maintain their bioactivity. We established an efficient ASK‐assisted method to express and purify plant F‐box proteins. The method meets a broad range of criteria required for the biochemical analysis or protein crystallization of plant F‐box proteins.     

Exploring the diversity of plant proteome

The tremendous functional, spatial, and temporal diversity of the plant proteome is regulated by multiple factors that continuously modify protein abundance, modifications, interactions, localization, and activity to meet the dynamic needs of plants. Dissecting the proteome complexity and its underlying genetic variation is attracting increasing research attention. Mass spectrometry (MS)-based proteomics has become a powerful approach in the global study of protein functions and their relationships on a systems level. Here, we review recent breakthroughs and strategies adopted to unravel the diversity of the proteome, with a specific focus on the methods used to analyze posttranslational modifications (PTMs), protein localization, and the organization of proteins into functional modules. We also consider PTM crosstalk and multiple PTMs temporally regulating the life cycle of proteins. Finally, we discuss recent quantitative studies using MS to measure protein turnover rates and examine future directions in the study of the plant proteome.     

Myofibrillar protein turnover. Synthesis of protein-bound 3-methylhistidine, actin, myosin heavy chain and aldolase in rat skeletal muscle in the fed and starved states.

The turnover of 3-methylhistidine (N tau-methylhistidine) and in some cases actin, myosin heavy chain and aldolase in skeletal muscle was measured in a number of experiments in growing and adult rats in the fed and overnight-starved states. In growing fed rats in three separate experiments, measurements of the methylation rate of protein-bound 3-methylhistidine by either [14C]- or [3H]-methyl-labelled S-adenosylmethionine show that 3-methylhistidine synthesis is slower than the overall rate of protein synthesis indicated by [14C]tyrosine incorporation. Values ranged from 36 to 51%. However, in one experiment with rapidly growing young fed rats, acute measurements over 1 h showed that 3-methylhistidine synthesis could be increased to the same rate as the overall rate. After overnight starvation in these rats, the steady-state synthesis rate of 3-methylhistidine was 38.8% of the overall rate. This was a similar value to that in adult non-growing rats, in which measurements of the relative labelling of 3-methylhistidine and histidine after a single injection of [14C]histidine indicated that 3-methylhistidine synthesis was 37% of the overall rate in the fed or overnight-starved state. According to measurements of actin, myosin heavy-chain and aldolase synthesis in the over-night-starved state with young rats, with a variety of precursors, slow turnover of 3-methylhistidine results from the specific slow turnover of actin, since turnover rates of myosin heavy chain, mixed protein and aldolase were 2.5, 3 and 3.4 times faster respectively. However, in the fed state synthesis rates of actin were increased disproportionately to give similar rates for all proteins. These results show that (a) 3-methylhistidine turnover in muscle is less than half the overall rate in both young and adult rats, (b) slow 3-methylhistidine turnover reflects the specifically slow turnover of actin compared with myosin heavy chain and other muscle proteins, and (c) during growth the synthesis rate of actin is particularly sensitive to the nutritional state and can be increased to a similar rate to that of other proteins.