Complete Mitochondrial Genome of the Medicinal Mushroom Ganoderma lucidum

Ganoderma lucidum is one of the well-known medicinal basidiomycetes worldwide. The mitochondrion, referred to as the second genome, is an organelle found in most eukaryotic cells and participates in critical cellular functions. Elucidating the structure and function of this genome is important to understand completely the genetic contents of G. lucidum. In this study, we assembled the mitochondrial genome of G. lucidum and analyzed the differential expressions of its encoded genes across three developmental stages. The mitochondrial genome is a typical circular DNA molecule of 60,630 bp with a GC content of 26.67%. Genome annotation identified genes that encode 15 conserved proteins, 27 tRNAs, small and large rRNAs, four homing endonucleases, and two hypothetical proteins. Except for genes encoding trnW and two hypothetical proteins, all genes were located on the positive strand. For the repeat structure analysis, eight forward, two inverted, and three tandem repeats were detected. A pair of fragments with a total length around 5.5 kb was found in both the nuclear and mitochondrial genomes, which suggests the possible transfer of DNA sequences between two genomes. RNA-Seq data for samples derived from three stages, namely, mycelia, primordia, and fruiting bodies, were mapped to the mitochondrial genome and qualified. The protein-coding genes were expressed higher in mycelia or primordial stages compared with those in the fruiting bodies. The rRNA abundances were significantly higher in all three stages. Two regions were transcribed but did not contain any identified protein or tRNA genes. Furthermore, three RNA-editing sites were detected. Genome synteny analysis showed that significant genome rearrangements occurred in the mitochondrial genomes. This study provides valuable information on the gene contents of the mitochondrial genome and their differential expressions at various developmental stages of G. lucidum. The results contribute to the understanding of the functions and evolution of fungal mitochondrial DNA.     

In-depth Characterization of the Cerebrospinal Fluid (CSF) Proteome Displayed Through the CSF Proteome Resource (CSF-PR)

In this study, the human cerebrospinal fluid (CSF) proteome was mapped using three different strategies prior to Orbitrap LC-MS/MS analysis: SDS-PAGE and mixed mode reversed phase-anion exchange for mapping the global CSF proteome, and hydrazide-based glycopeptide capture for mapping glycopeptides. A maximal protein set of 3081 proteins (28,811 peptide sequences) was identified, of which 520 were identified as glycoproteins from the glycopeptide enrichment strategy, including 1121 glycopeptides and their glycosylation sites. To our knowledge, this is the largest number of identified proteins and glycopeptides reported for CSF, including 417 glycosylation sites not previously reported. From parallel plasma samples, we identified 1050 proteins (9739 peptide sequences). An overlap of 877 proteins was found between the two body fluids, whereas 2204 proteins were identified only in CSF and 173 only in plasma. All mapping results are freely available via the new CSF Proteome Resource (http://probe.uib.no/csf-pr), which can be used to navigate the CSF proteome and help guide the selection of signature peptides in targeted quantitative proteomics.Cerebrospinal fluid (CSF)¹ surrounds and supports the central nervous system (CNS), including the ventricles and subarachnoid space (1). About 80% of the total protein amount in CSF derives from size-dependent filtration of blood across the blood-brain barrier (BBB), and the rest originate from drainage of interstitial fluid from the CNS (2–4). Because CSF is in direct contact with the CNS, it should be a promising source for finding biomarkers for diseases in the CNS (5).Mapping studies characterizing the human CSF proteome and peptidome has previously been carried out using various experimental designs, including both healthy and disease-affected individuals (5–16). A total of 2630 proteins were detected in normal CSF by immunoaffinity depletion of high abundant proteins followed by strong cation exchange fractionation and LC-MS (5), whereas proteome and peptidome analyses of human CSF (collected for diagnostic purposes and turned out normal) by gel separation and trypsin digestion followed by LC-MS analysis have shown 798 proteins and 563 peptide products (derived from 91 precursor proteins) (6). In another publication, Pan et al. combined several proteomics studies in CSF from both normal subjects and subjects with neurological diseases and created a dataset of 2594 identified proteins (16). But in general, the availability and usefulness of published data from proteome mapping experiments is scarce, and the format of the data often makes searching and comparison across datasets difficult. Thus, organizing the data in online databases would greatly benefit the scientific community by making the data more accessible and easier to query. Current online databases containing MS data for CSF include the Sys-BodyFluid, with a total of 1286 CSF proteins from six studies (17). The proteome identifications database (PRIDE) (18) includes 19 studies on human CSF, but none reporting more than 103 identified proteins.Glycosylation is one of the most common post-translational modifications (PTMs), and many known clinical biomarkers as well as therapeutic targets are glycoproteins (19–25). Furthermore, glycosylation plays important roles in cell communication, signaling, aging, and cell adhesion (26, 27). Nevertheless, there are few studies on glycoprotein identification in CSF. One study identified 216 glycoproteins in CSF using both lectin affinity and hydrazide chemistry (8), and another reported 36 N-linked and 44 O-linked glycosylation sites, from 23 and 22 glycoproteins respectively, by enriching for sialic-acid containing glycopeptides (28).Considering the sparse information about the CSF proteome available in public repositories, we have combined several proteomics approaches to create a map of the global CSF proteome, the CSF glycoproteome, and the respective plasma proteome from a pool of 21 (20 for the plasma pool) neurologically healthy individuals. The large amount of data generated through these four datasets (with linked and complementary information) would not easily be accessible through existing repositories. We therefore developed the open access CSF Proteome Resource (CSF-PR, www.probe.uib.no/csf-pr), an online database including the detailed data from the four different proteomics experiments described in this study. CSF-PR will be particularly useful in guiding the selection of appropriate signature peptides for the development of targeted CSF protein assays.     

Enhancement of Ganoderic Acid Accumulation by Overexpression of an N-Terminally Truncated 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase Gene in the Basidiomycete Ganoderma lucidum

Ganoderic acids produced by Ganoderma lucidum, a well-known traditional Chinese medicinal mushroom, exhibit antitumor and antimetastasis activities. Genetic modification of G. lucidum is difficult but critical for the enhancement of cellular accumulation of ganoderic acids. In this study, a homologous genetic transformation system for G. lucidum was developed for the first time using mutated sdhB, encoding the iron-sulfur protein subunit of succinate dehydrogenase, as a selection marker. The truncated G. lucidum gene encoding the catalytic domain of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) was overexpressed by using the Agrobacterium tumefaciens-mediated transformation system. The results showed that the mutated sdhB successfully conferred carboxin resistance upon transformation. Most of the integrated transfer DNA (T-DNA) appeared as a single copy in the genome. Moreover, deregulated constitutive overexpression of the HMGR gene led to a 2-fold increase in ganoderic acid content. It also increased the accumulation of intermediates (squalene and lanosterol) and the upregulation of downstream genes such as those of farnesyl pyrophosphate synthase, squalene synthase, and lanosterol synthase. This study demonstrates that transgenic basidiomycete G. lucidum is a promising system to achieve metabolic engineering of the ganoderic acid pathway.     

Intermembrane Space Proteome of Yeast Mitochondria

The intermembrane space (IMS) represents the smallest subcompartment of mitochondria. Nevertheless, it plays important roles in the transport and modification of proteins, lipids, and metal ions and in the regulation and assembly of the respiratory chain complexes. Moreover, it is involved in many redox processes and coordinates key steps in programmed cell death. A comprehensive profiling of IMS proteins has not been performed so far. We have established a method that uses the proapoptotic protein Bax to release IMS proteins from isolated mitochondria, and we profiled the protein composition of this compartment. Using stable isotope-labeled mitochondria from Saccharomyces cerevisiae, we were able to measure specific Bax-dependent protein release and distinguish between quantitatively released IMS proteins and the background efflux of matrix proteins. From the known 31 soluble IMS proteins, 29 proteins were reproducibly identified, corresponding to a coverage of >90%. In addition, we found 20 novel intermembrane space proteins, out of which 10 had not been localized to mitochondria before. Many of these novel IMS proteins have unknown functions or have been reported to play a role in redox regulation. We confirmed IMS localization for 15 proteins using in organello import, protease accessibility upon osmotic swelling, and Bax-release assays. Moreover, we identified two novel mitochondrial proteins, Ymr244c-a (Coa6) and Ybl107c (Mic23), as substrates of the MIA import pathway that have unusual cysteine motifs and found the protein phosphatase Ptc5 to be a novel substrate of the inner membrane protease (IMP). For Coa6 we discovered a role as a novel assembly factor of the cytochrome c oxidase complex. We present here the first and comprehensive proteome of IMS proteins of yeast mitochondria with 51 proteins in total. The IMS proteome will serve as a valuable source for further studies on the role of the IMS in cell life and death.Mitochondria are double-membrane-bound organelles that fulfill a multitude of important cellular functions. Proteomic analysis of purified mitochondria revealed that they contain approximately 1000 (yeast) to 1500 (human) different proteins (1–3). However, the distribution of these proteins among the four mitochondrial subcompartments (outer membrane, inner membrane, matrix, and intermembrane space) has been only marginally studied through global approaches. This is attributed to the high complexity of purifying submitochondrial fractions to a grade suitable for proteomic analysis. The best-studied submitochondrial proteomes comprise the outer membranes of S. cerevisae, N. crassa, and A. thaliana (4–6). The mitochondrial intermembrane space (IMS)¹ represents a highly interesting compartment for several reasons: it provides a redox active space that promotes oxidation of cysteine residues similar to the endoplasmic reticulum and the bacterial periplasm, but unlike cytosol, nucleus, or the mitochondrial matrix where the presence of thioredoxins or glutaredoxins prevents the risk of unwanted cysteine oxidation (7, 8). Furthermore in higher eukaryotes IMS proteins are released into the cytosol upon apoptotic induction, which triggers the activation of a cell-killing protease activation cascade (9, 10). The IMS can also exchange proteins, lipids, metal ions, and various metabolites with other cellular compartments, allowing mitochondrial metabolism to adapt to cellular homeostasis. In particular, the biogenesis and activity of the respiratory chain were shown to be controlled by various proteins of the IMS (11–13). Most of the currently known IMS proteins are soluble proteins; however, some inner membrane proteins have been annotated as IMS proteins as well, such as proteins that are peripherally attached to the inner membrane or membrane proteins that expose enzyme activity toward the IMS (8).All IMS proteins are encoded in the nuclear DNA and have to be imported after translation in the cytosol (14–19). Two main pathways are known to mediate the import and sorting of proteins into the IMS. One class of proteins contains bipartite presequences that consist of a matrix targeting signal and a hydrophobic sorting signal. These signals arrest the incoming preprotein at the inner membrane translocase TIM23. After insertion into the inner membrane, the soluble, mature protein can be released into the IMS by the inner membrane protease (IMP) (20–22). The second class of IMS proteins possesses characteristic cysteine motifs that typically are either twin CX₉C or twin CX₃C motifs (23, 24). Upon translocation across the outer membrane via the TOM complex, disulfide bonds are formed within the preproteins, which traps them in the IMS. Disulfide bond formation is mediated by the MIA machinery, which consists of the inner-membrane-anchored Mia40 and the soluble IMS protein Erv1 (25–28).The release of cytochrome c from the IMS upon binding and insertion of Bax at the outer membrane is a hallmark of programmed cell death. Although Bax is found only in higher eukaryotes, it was shown that recombinant mammalian Bax induces the release of cytochrome c upon incubation with isolated yeast mitochondria (29, 30). Furthermore, we found that not only cytochrome c but also other soluble IMS proteins are released from Bax-treated yeast mitochondria, whereas soluble matrix proteins largely remain within the organelle (30).We used this apparently conserved mechanism to systematically profile the protein composition of the yeast mitochondrial IMS by employing an experimental approach based on stable isotope labeling, which allowed for the specific identification of Bax-dependent protein release. Almost the entire set of known soluble IMS proteins was identified, and 20 additional, novel soluble IMS proteins were found. We confirmed IMS localization for 15 proteins through biochemical assays. Among these proteins, we identified novel proteins that fall into several classes: (i) those that are involved in maintaining protein redox homeostasis (thioredoxins, thioredoxin reductases, or thiol peroxidases), (ii) those that undergo proteolytic processing by IMP (Ptc5), (iii) those that utilize the MIA pathway for their import (Mic23 and Coa6), and (iv) those that play a role in the assembly of cytochrome c oxidase (Coa6).     

The Proteome Response to Amyloid Protein Expression In Vivo

Protein misfolding disorders such as Alzheimer, Parkinson and transthyretin amyloidosis are characterized by the formation of protein amyloid deposits. Although the nature and location of the aggregated proteins varies between different diseases, they all share similar molecular pathways of protein unfolding, aggregation and amyloid deposition. Most effects of these proteins are likely to occur at the proteome level, a virtually unexplored reality. To investigate the effects of an amyloid protein expression on the cellular proteome, we created a yeast expression system using human transthyretin (TTR) as a model amyloidogenic protein. We used Saccharomyces cerevisiae, a living test tube, to express native TTR (non-amyloidogenic) and the amyloidogenic TTR variant L55P, the later forming aggregates when expressed in yeast. Differential proteome changes were quantitatively analyzed by 2D-differential in gel electrophoresis (2D-DIGE). We show that the expression of the amyloidogenic TTR-L55P causes a metabolic shift towards energy production, increased superoxide dismutase expression as well as of several molecular chaperones involved in protein refolding. Among these chaperones, members of the HSP70 family and the peptidyl-prolyl-cis-trans isomerase (PPIase) were identified. The latter is highly relevant considering that it was previously found to be a TTR interacting partner in the plasma of ATTR patients but not in healthy or asymptomatic subjects. The small ubiquitin-like modifier (SUMO) expression is also increased. Our findings suggest that refolding and degradation pathways are activated, causing an increased demand of energetic resources, thus the metabolic shift. Additionally, oxidative stress appears to be a consequence of the amyloidogenic process, posing an enhanced threat to cell survival.     

Dynamic Proteome Response of Pseudomonas aeruginosa to Tobramycin Antibiotic Treatment

Genetically susceptible bacteria become antibiotic tolerant during chronic infections, and the mechanisms responsible are poorly understood. One factor that may contribute to differential sensitivity in vitro and in vivo is differences in the time-dependent tobramycin concentration profile experienced by the bacteria. Here, we examine the proteome response induced by subinhibitory concentrations of tobramycin in Pseudomonas aeruginosa cells grown under planktonic conditions. These efforts revealed increased levels of heat shock proteins and proteases were present at higher dosage treatments (0.5 and 1 μg/ml), while less dramatic at 0.1 μg/ml dosage. In contrast, many metabolic enzymes were significantly induced by lower dosages (0.1 and 0.5 μg/ml) but not at 1 μg/ml dosage. Time course proteome analysis further revealed that the increase of heat shock proteins and proteases was most rapid from 15 min to 60 min, and the increased levels sustained till 6 h (last time point tested). Heat shock protein IbpA exhibited the greatest induction by tobramycin, up to 90-fold. Nevertheless, deletion of ibpA did not enhance sensitivity to tobramycin. It seemed possible that the absence of sensitization could be due to redundant functioning of IbpA with other proteins that protect cells from tobramycin. Indeed, inactivation of two heat shock chaperones/proteases in addition to ibpA in double mutants (ibpA/clpB, ibpA/PA0779 and ibpA/hslV) did increase tobramycin sensitivity. Collectively, these results demonstrate the time- and concentration-dependent nature of the P. aeruginosa proteome response to tobramycin and that proteome modulation and protein redundancy are protective mechanisms to help bacteria resist antibiotic treatments.The opportunistic pathogen Pseudomonas aeruginosa is ubiquitous in the natural environment and causes human infections (1). P. aeruginosa can metabolize various carbon and nitrogen compounds and persists under nutrient-poor and hostile growth environments (2, 3). One example is P. aeruginosa pulmonary infection of cystic fibrosis (CF) patients. Despite stress induced by host defenses and high concentrations of antibiotics, P. aeruginosa cells are able to persistently colonize CF airways (4).The aminoglycoside tobramycin is a front-line drug currently used in the treatment of P. aeruginosa in CF and other diseases. It is supplied in the forms of inhaled solution (TOBI) and intravenous injection. The tobramycin concentrations in airways after 300-mg dosage TOBI inhalation can reach 1,000 μg per g of sputum (5, 6). This concentration is in the range of 10 to 1,000 times of the minimal inhibitory concentration (MIC) for P. aeruginosa clinical isolates tested ex vivo (6). However, even with such high tobramycin concentrations, chronic P. aeruginosa infections are rarely eradicated (6). This is true even when the infecting bacteria are antibiotic sensitive, as is the case early in disease (7).One possible reason for P. aeruginosa persistence in vivo could relate to the time dependence of local concentrations of tobramycin experienced by P. aeruginosa in CF patient airways. Many factors, including inflammatory responses, blood and lymphatic circulations, and air flow distribution (for inhaled antibiotics), can alter the local antibiotic concentrations. In addition, P. aeruginosa cells can form biofilms in CF lungs and other infection sites (8), and biofilm exopolysaccharide layers may slow the diffusion of tobramycin (9, 10). P. aeruginosa cells in the inner layers of biofilms may experience lower concentrations and more gradual increase of tobramycin levels than those in outer layers (10, 11). Furthermore, even if final tobramycin concentration levels inside the biofilm eventually grow to match the highest levels experienced elsewhere, bacteria in these inner regions have experienced a slower increase, during which time proteome levels could be altered to promote the “adapted resistant state” (12). Adaptive resistance can also be induced in planktonic (free-living) P. aeruginosa (13, 14), and conventional MIC assays are not designed to measure this.Once induced, the adaptive resistance confers bacteria higher resistance to antibiotic treatments (13, 14) and is associated with decreased clinical antibiotic treatment efficacy (15). Interestingly, the adaptive resistance is time dependent and reversible. Typical adaptive resistance was observed starting 1 h after antibiotic exposure, and the drug susceptibility was regained after 36 h intervals (14, 15). Thus, adaptive resistance mechanisms may contribute in part to the disparity of in vivo persistence and ex vivo susceptibility to antibiotics in MIC tests.As an initial step toward defining adaptive resistance mechanisms, we investigated the time- and concentration-dependence of P. aeruginosa proteome response to tobramycin in planktonic conditions. Since the most effective protective responses may operate before killing begins and the rate of change of drug levels is likely to depend on ambient conditions, we studied bacteria exposed to low, subinhibitory levels of tobramycin (0.1, 0.5, and 1.0 μg/ml) at a range of time points (15, 60, 120, and 360 min) after exposure. The candidate proteome marker of P. aeruginosa for tobramycin response, heat shock protein IbpA, was further investigated with genetic mutagenesis and MIC assays.     

Quantification of Extracellular Matrix Proteins from a Rat Lung Scaffold to Provide a Molecular Readout for Tissue Engineering

The use of extracellular matrix (ECM)¹ scaffolds, derived from decellularized tissues for engineered organ generation, holds enormous potential in the field of regenerative medicine. To support organ engineering efforts, we developed a targeted proteomics method to extract and quantify extracellular matrix components from tissues. Our method provides more complete and accurate protein characterization than traditional approaches. This is accomplished through the analysis of both the chaotrope-soluble and -insoluble protein fractions and using recombinantly generated stable isotope labeled peptides for endogenous protein quantification. Using this approach, we have generated 74 peptides, representing 56 proteins to quantify protein in native (nondecellularized) and decellularized lung matrices. We have focused on proteins of the ECM and additional intracellular proteins that are challenging to remove during the decellularization procedure. Results indicate that the acellular lung scaffold is predominantly composed of structural collagens, with the majority of these proteins found in the insoluble ECM, a fraction that is often discarded using widely accepted proteomic methods. The decellularization procedure removes over 98% of intracellular proteins evaluated and retains, to varying degrees, proteoglycans and glycoproteins of the ECM. Accurate characterization of ECM proteins from tissue samples will help advance organ engineering efforts by generating a molecular readout that can be correlated with functional outcome to drive the next generation of engineered organs.Organ transplantation is an established, lifesaving therapy for patients with chronic end-stage diseases. However, transplantation as a therapeutic option is limited by availability of suitable donor organs (1). Although advancements in surgical techniques, such as successful implementation of bilateral lung transplants and improved immunosuppressant treatments, have led to more successful outcomes in recent years, the percentage of people that die while on the transplant wait list has increased (2, 3). One attractive approach to meet this demand is the in vitro generation of organs using decellularized tissues as scaffolds for recellularization. For complex organs such as the lung, these tissue scaffolds can be derived from a donor organ that would have otherwise been unfit for transplantation. This whole organ scaffold can be recellularized using a patient''s own primary or stem-derived cells, thus eliminating many issues related to graft/host incompatibility. This approach was recently used to generate lungs that, when implanted in rat recipients, allowed for gas exchange (4, 5). However, examination of the lung indicated leakage of erythrocytes into the alveolar space, indicating a compromised capillary-endothelial barrier. These exciting results highlighted the potential of the method for organ transplantation but also the need for improved molecular readouts to guide engineering efforts.Efficient reseeding of decellularized scaffolds has been shown to be dependent on retaining native ECM structural integrity and elasticity (6). Local variations in expression of abundant proteins in the ECM scaffolding (collagens, laminins, fibronectins) have been correlated to variance in cell repopulation and subsequent proliferation (7). It is thought that retaining specific ECM components and architectures may allow cells to be directed back to a tissue-specific niche during reseeding and that small changes in abundance of these molecular cues can drastically affect the recellularization process (8). Current methods used to characterize the protein composition of native and acellular tissues involve antibody- or dye-based staining, hydroxyproline assays assessing collagen content, or relative quantification of proteins by liquid chromatography tandem mass spectrometry (LC-MS/MS) (9, 10). All of these methods either fall short in specificity, accurate quantification, or both. A more complete and accurate method for protein characterization would provide a valuable tool for tissue engineering efforts, while shedding light on the possible molecular mechanisms resulting in cell seeding variability and alterations in mechanical properties of engineered lung tissues.Current relative quantification strategies (iTRAQ, Spectral Counting, dimethyl labeling, others) (11–15) perform well when the majority of protein in samples does not change, there are approximately equal increases and decreases in protein levels, or in cases where proteins that are known not to change in abundance can be used for normalization. However, normalization steps often employed have the potential to introduce experimental bias (16). The decellularization process differentially removes and enriches proteins in the ECM scaffolding, depleting some proteins with high efficiency while leaving others mostly intact. This makes relative comparisons between native and decellularized lung challenging. Although strategies can be employed in an attempt to normalize data (17), there is a distinct advantage to quantification methods using stable isotope labeled (SIL) peptides in this application. Here, we developed ECM targeted, isotopically labeled peptides using the QconCAT approach first described by Beynon et al. (18). SIL quantification allows for intra- and intersample comparison of heterogeneous tissues, such as native organs and decellularized scaffolds, with high accuracy and precision.The ECM is largely responsible for defining the biomechanical properties of organs. Maintaining structural rigidity and native microarchitecture through the decellularization process makes an acellular organ a good candidate to serve as a tissue scaffold (19, 20). These same characteristics are a central reason why the ECM is challenging to characterize using common bottom-up proteomics approaches (21). Currently accepted and widely used digestion methods require proteins to be solubilized for bottom-up proteomic analysis (22). Recent papers have reported characterization of the ECM fraction from tissues through the use of strong chaotropes (11, 21, 23–27) or cellular fractionation followed by strong detergent (10, 28, 29). However, in our experience, these protocols invariably yield various sizes of an insoluble protein-containing pellet when applied to a variety of tissue samples (heart, lung, and mammary gland). On one end of the spectrum, methods utilizing deglycosylation and enzymatic digestions for clarification of partial solubilized protein slurries yields good ECM coverage with a high number of spectral matches for collagen alpha-1(I), a highly abundant ECM protein in lung (28). On the other end of the spectrum, methods using only detergents or chaotropes for solubilization result in protein pellets that are generally removed prior to LC-MS/MS analysis. These pellets often contained a majority of fibrillar proteins, resulting in quantitative errors. Consistent with this finding, several of these studies characterizing tissue engineered lungs do not report the identification of collagen alpha-1(I) (8, 10, 30). We believe these observations result from a failure to solubilize and enzymatically digest insoluble ECM proteins. To this end, we explored the use of chemical digestion of the insoluble pellet to improve coverage of the ECM proteome from tissue. This method has been used to quantify protein levels from native and decellularized lungs to determine decellularization specificity and efficiency. The accurate characterization of ECM proteins from lung samples should advance tissue engineering efforts by yielding a readout that can be correlated with functional outcome to drive further development.     

Metabolic Labeling Reveals Proteome Dynamics of Mouse Mitochondria

Mitochondrial dysfunction is associated with many human diseases. Mitochondrial damage is exacerbated by inadequate protein quality control and often further contributes to pathogenesis. The maintenance of mitochondrial functions requires a delicate balance of continuous protein synthesis and degradation, i.e. protein turnover. To understand mitochondrial protein dynamics in vivo, we designed a metabolic heavy water (²H₂O) labeling strategy customized to examine individual protein turnover in the mitochondria in a systematic fashion. Mice were fed with ²H₂O at a minimal level (<5% body water) without physiological impacts. Mitochondrial proteins were analyzed from 9 mice at each of the 13 time points between 0 and 90 days (d) of labeling. A novel multiparameter fitting approach computationally determined the normalized peak areas of peptide mass isotopomers at initial and steady-state time points and permitted the protein half-life to be determined without plateau-level ²H incorporation. We characterized the turnover rates of 458 proteins in mouse cardiac and hepatic mitochondria and found median turnover rates of 0.0402 d⁻¹ and 0.163 d⁻¹, respectively, corresponding to median half-lives of 17.2 d and 4.26 d. Mitochondria in the heart and those in the liver exhibited distinct turnover kinetics, with limited synchronization within functional clusters. We observed considerable interprotein differences in turnover rates in both organs, with half-lives spanning from hours to months (∼60 d). Our proteomics platform demonstrates the first large-scale analysis of mitochondrial protein turnover rates in vivo, with potential applications in translational research.Mitochondrial dysfunctions are observed in disorders such as neurodegeneration, cardiovascular diseases, and aging (1–3). It is postulated that the failure to contain or replenish mitochondrial proteins damaged by reactive oxygen species directly underlies many pathological phenotypes (4). The development of effective treatments for these diseases therefore relies on understanding the molecular basis of protein dynamics. Outstanding questions are how the processes of mitochondrial proteome dynamics are regulated in different systems, and how their perturbations could progress to pathological remodeling of the organelle. Thus far, quantitative proteomics efforts have been predominated by steady-state measurements, which often provide fragmentary snapshots of the proteome that are difficult to comprehend in the context of other cellular events.To further understand mitochondrial dynamics in vivo, we examined the turnover rates of individual heart and liver mitochondrial proteins on a proteome scale. Both the liver and the heart contain large numbers of mitochondria, but cardiac and hepatic mitochondria differ in their protein composition, oxygen consumption, substrate utilization, and disease manifestation. However, these differences are often interpreted only by protein compositions and steady-state abundance, without the consideration of protein kinetics in the temporal dimension. Abnormal protein kinetics may indicate dysfunctions in protein quality control, the accumulation of damaged proteins, misfolding, or other proteinopathies. Protein dynamics itself is an important intrinsic property of the proteome, the disruption of which could be causal of cellular etiologies.At minimum, a kinetic definition of the proteome requires knowledge of the rate at which individual proteins are being replaced. Isotope tracers are particularly useful for tracking such continual renewal of the proteome in living systems, because they allow differentiation between preexisting and newly synthesized proteins (5). Among the available stable isotope precursors, heavy water (²H₂O) labeling offers several advantages with respect to safety, labeling kinetics, and cost (6, 7). First, ²H₂O administration to animals and humans at low enrichment levels is safe for months or even years (8). Second, maintaining constant ²H enrichment levels in body water following the initial intake of ²H₂O is easily achieved, because administrated ²H₂O rapidly equilibrates over all tissues but decays slowly (9, 10). Third, ²H₂O labeling is more cost effective than other stable isotope labeling methods. Importantly, ²H₂O intake induces universal ²H incorporation into biomolecules. Systematic insights into protein turnover in vivo could therefore be correlated to that of nucleic acids, carbohydrates, or lipids, enabling broad applications for this technology in studying mammalian systems, including humans.A variety of methodologies have been developed to analyze the extent of ²H incorporation in proteins following ²H₂O labeling, including GC-MS measurements of hydrolyzed target proteins (11–14) and peptide analysis in MALDI-TOF MS (15) and LC-MS (16, 17). More recently, Price et al. described an approach for measuring protein turnover by calculating the theoretical number of ²H-labeling sites on a peptide sequence (18) and reported the turnover rates of ∼100 human plasma proteins. Here we describe another novel strategy to determine protein turnover rates on a proteomic scale using ²H₂O labeling. By computing the parameters needed to deduce fractional protein synthesis using software we developed, we were able to obtain protein half-life data without relying on the asymptotic isotopic abundance of peptide ions. Our approach also has the unique benefit of automating all steps of isotopomer quantification and postcollection data analysis, and it does not require knowledge of the exact precursor enrichment or labeling sites of peptides. We observed diverse kinetics from 458 liver and heart mitochondrial proteins that inform essential characteristics of mitochondrial dynamics and intragenomic differences between the two organs.     

An In-solution Ultrasonication-assisted Digestion Method for Improved Extracellular Matrix Proteome Coverage

Epithelial cell behavior is coordinated by the composition of the surrounding extracellular matrix (ECM); thus ECM protein identification is critical for understanding normal biology and disease states. Proteomic analyses of ECM proteins have been hindered by the insoluble and digestion-resistant nature of ECM. Here we explore the utility of combining rapid ultrasonication- and surfactant-assisted digestion for the detailed proteomics analysis of ECM samples. When compared with traditional overnight digestion, this optimized method dramatically improved the sequence coverage for collagen I, revealed the presence of hundreds of previously unidentified proteins in Matrigel, and identified a protein profile for ECM isolated from rat mammary glands that was substantially different from that found in Matrigel. In a three-dimensional culture assay to investigate epithelial cell-ECM interactions, mammary epithelial cells were found to undergo extensive branching morphogenesis when plated with mammary gland-derived matrix in comparison with Matrigel. Cumulatively these data highlight the tissue-specific nature of ECM composition and function and underscore the need for optimized techniques, such as those described here, for the proteomics characterization of ECM samples.Extracellular matrix (ECM)¹ is a critical component of the tissue microenvironment. ECM plays a pivotal role in embryonic stem cell development and differentiation (1, 2) as well as many physiological (3) and pathological processes, including cancer progression (4, 5). Cell regulation by ECM has been studied with high frequency in recent years (7, 8). However, our ability to globally characterize ECM composition both in vitro and in vivo has been severely limited because of several unique attributes of ECM proteins such as high molecular weight glycans and the presence of covalent protein cross-links (6, 9, 10). Traditional proteomics approaches have proven to be ineffective for the identification of ECM proteins as demonstrated by the fact that collagens, despite being the most abundant protein in mammals, are significantly underrepresented in tissue-based proteomics data sets.Ultrasonication has long been used for the digestion of bioorganic materials to allow for maximal and reproducible extraction and hence the accurate identification of small molecule and inorganic analytes (11). More recently, Capelo et al. (12) have used ultrasonic energy to catalyze tryptic digestion of proteins for subsequent mass spectrometry-based identification. Here we sought to determine whether this method could be optimized to prepare ECM samples for mass spectrometry-based analysis. For method development, we used rat tail collagen as a representative ECM protein for which current proteomics approaches have proven relatively unsuccessful. Type I collagen is defined as a right-handed triple helix heterotrimer comprising two identical α1 chains and one α2 chain that form a fibrillar network (6). The physical properties of the triple helical structure render the protein resistant to proteasch as trypsin (9). In this work, we focused our efforts on developing a digestion approach that improves our ability to perform proteomics analysis on a type I collagen preparation and then used this method to identify the protein composition of EHS murine chondrosarcoma matrix (10), herein referred to as Matrigel, and a matrix preparation from rat mammary tissue.In this study, we developed a digestion approach suitable for a two-dimensional liquid chromatography-tandem mass spectrometry-based analysis of ECM proteins. Our digestion approach involves three cycles of ultrasonication for rapid initial trypsin digestion followed by overnight digestion using an acid-labile surfactant. This approach resulted in significant improvement in collagen peptide identification and the identification of numerous ECM proteins previously uncharacterized in Matrigel and in mammary tissue. The application of our ECM-optimized ultrasonic assisted trypsin digestion method is anticipated to significantly advance the identification of tissue- and disease state-specific ECM proteins.     

Chlamydia trachomatis Infection Leads to Defined Alterations to the Lipid Droplet Proteome in Epithelial Cells

The obligate intracellular bacterium Chlamydia trachomatis is a major human pathogen and a main cause of genital and ocular diseases. During its intracellular cycle, C. trachomatis replicates inside a membrane-bound vacuole termed an “inclusion”. Acquisition of lipids (and other nutrients) from the host cell is a critical step in chlamydial replication. Lipid droplets (LD) are ubiquitous, ER-derived neutral lipid-rich storage organelles surrounded by a phospholipids monolayer and associated proteins. Previous studies have shown that LDs accumulate at the periphery of, and eventually translocate into, the chlamydial inclusion. These observations point out to Chlamydia-mediated manipulation of LDs in infected cells, which may impact the function and thereby the protein composition of these organelles. By means of a label-free quantitative mass spectrometry approach we found that the LD proteome is modified in the context of C. trachomatis infection. We determined that LDs isolated from C. trachomatis-infected cells were enriched in proteins related to lipid metabolism, biosynthesis and LD-specific functions. Interestingly, consistent with the observation that LDs intimately associate with the inclusion, a subset of inclusion membrane proteins co-purified with LD protein extracts. Finally, genetic ablation of LDs negatively affected generation of C. trachomatis infectious progeny, consistent with a role for LD biogenesis in optimal chlamydial growth.     

Comparative Proteome Analysis of Multi-Layer Cocoon of the Silkworm,Bombyx mori

Bombyx mori cocoon has a multi-layer structure that provides optimal protection for silkworm pupa. Research on the mechanical properties of the multi-layer structure revealed structure-property relationships of the cocoon. Here, we investigated the protein components of the B. mori cocoon in terms of its multi-layer structure. Liquid chromatography-tandem mass spectrometry identified 286 proteins from the multiple cocoon layers. In addition to fibroins and sericins, we identified abundant protease inhibitors, seroins and proteins of unknown function. By comparing protein abundance across layers, we found that the outermost layer contained more sericin1 and protease inhibitors and the innermost layer had more seroin1. As many as 36 protease inhibitors were identified in cocoons, showing efficient inhibitory activities against a fungal protease. Thus, we propose that more abundant protease inhibitors in the outer cocoon layers may provide better protection for the cocoon. This study increases our understanding of the multi-layer mechanism of cocoons, and helps clarify the biological characteristics of cocoons. The data have been deposited to the ProteomeXchange with identifier PXD001469.     

Prokayrotic Ubiquitin-Like Protein (Pup) Proteome of Mycobacterium tuberculosis

Prokaryotic ubiquitin-like protein (Pup) in Mycobacterium tuberculosis (Mtb) is the first known post-translational small protein modifier in prokaryotes, and targets several proteins for degradation by a bacterial proteasome in a manner akin to ubiquitin (Ub) mediated proteolysis in eukaryotes. To determine the extent of pupylation in Mtb, we used tandem affinity purification to identify its “pupylome”. Mass spectrometry identified 55 out of 604 purified proteins with confirmed pupylation sites. Forty-four proteins, including those with and without identified pupylation sites, were tested as substrates of proteolysis in Mtb. Under steady state conditions, the majority of the test proteins did not accumulate in degradation mutants, suggesting not all targets of pupylation are necessarily substrates of the proteasome under steady state conditions. Four proteins implicated in Mtb pathogenesis, Icl (isocitrate lyase), Ino1 (inositol-1-phosphate synthase), MtrA (Mtb response regulator A) and PhoP (phosphate response regulator P), showed altered levels in degradation defective Mtb. Icl, Ino1 and MtrA accumulated in Mtb degradation mutants, suggesting these proteins are targeted to the proteasome. Unexpectedly, PhoP was present in wild type Mtb but undetectable in the degradation mutants. Taken together, these data demonstrate that pupylation regulates numerous proteins in Mtb and may not always lead to degradation.