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).     

Matrix Assisted Ionization Vacuum (MAIV), a New Ionization Method for Biological Materials Analysis Using Mass Spectrometry

The introduction of electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) for the mass spectrometric analysis of peptides and proteins had a dramatic impact on biological science. We now report that a wide variety of compounds, including peptides, proteins, and protein complexes, are transported directly from a solid-state small molecule matrix to gas-phase ions when placed into the vacuum of a mass spectrometer without the use of high voltage, a laser, or added heat. This ionization process produces ions having charge states similar to ESI, making the method applicable for high performance mass spectrometers designed for atmospheric pressure ionization. We demonstrate highly sensitive ionization using intermediate pressure MALDI and modified ESI sources. This matrix and vacuum assisted soft ionization method is suitable for the direct surface analysis of biological materials, including tissue, via mass spectrometry.The conversion of large and nonvolatile compounds such as proteins into gas-phase ions is of immense fundamental and practical importance. The 2002 Nobel Prize in Chemistry was awarded for the accomplishment of this conversion via electrospray ionization (ESI)¹ (1) and matrix-assisted laser desorption/ionization (MALDI) (2) interfaced with mass spectrometry (MS) to obtain the molecular weights of proteins with high accuracy. These methods employ high voltage or a laser to form gaseous analyte ions from a wide variety of compounds in solution or a solid matrix, respectively.MALDI interfaced with a time-of-flight (TOF) mass spectrometer produces gas-phase analyte ions in vacuum and is the method of choice for the molecular imaging of biological surfaces. Ionization in vacuum provides excellent ion transmission (3), as well as good spatial resolution achieved using a focused laser beam. However, the analysis of protein complexes is very challenging with MALDI, requiring strategies such as first-shot phenomena (4) and chemical crosslinking (5). The necessity of a laser also makes MALDI less soft than ESI and produces background ions, which can hinder the analysis of small molecules (6, 7). MALDI is also of limited utility on high performance mass-to-charge (m/z) analyzers because of mass range issues related to the formation of singly charged ions, which also produce few fragment ions for structural characterization (8).Multiple charged ions produced directly from solution in ESI bring the m/z ratio within the range of high performance mass spectrometers, allowing the analysis of high-mass compounds. These instruments have advanced features for structural characterization, such as ion mobility spectrometry (IMS) for gas-phase separations (9–11), ultra-high mass resolution and mass accuracy (12–14), and advanced fragmentation such as electron transfer dissociation (ETD) (13, 14). However, ESI is limited for surface characterization, requiring approaches such as desorption-ESI (15) and laser ablation ESI (16), ionization methods that produce multiply charged ions but are not compatible with analyses of larger proteins or fragile complexes.A softer ionization approach is needed in order to observe fragile molecules and molecular complexes in living organisms at low levels directly from tissue and cell cultures, without extensive sample preparation, while retaining spatial information. Ideally, this approach would be compatible with mass spectrometers having advanced capabilities to aid structural characterization directly from surfaces. The new ionization method described here, in which molecules are transferred from solid-phase to gas-phase ions through the simple exposure of a material of interest in a suitable matrix to vacuum, is an advance toward this goal and is of fundamental interest.     

A Small Conserved Domain in the Yeast Spa2p Is Necessary and Sufficient for Its Polarized Localization

SPA2 encodes a yeast protein that is one of the first proteins to localize to sites of polarized growth, such as the shmoo tip and the incipient bud. The dynamics and requirements for Spa2p localization in living cells are examined using Spa2p green fluorescent protein fusions. Spa2p localizes to one edge of unbudded cells and subsequently is observable in the bud tip. Finally, during cytokinesis Spa2p is present as a ring at the mother–daughter bud neck. The bud emergence mutants bem1 and bem2 and mutants defective in the septins do not affect Spa2p localization to the bud tip. Strikingly, a small domain of Spa2p comprised of 150 amino acids is necessary and sufficient for localization to sites of polarized growth. This localization domain and the amino terminus of Spa2p are essential for its function in mating. Searching the yeast genome database revealed a previously uncharacterized protein which we name, Sph1p (Spa2p homolog), with significant homology to the localization domain and amino terminus of Spa2p. This protein also localizes to sites of polarized growth in budding and mating cells. SPH1, which is similar to SPA2, is required for bipolar budding and plays a role in shmoo formation. Overexpression of either Spa2p or Sph1p can block the localization of either protein fused to green fluorescent protein, suggesting that both Spa2p and Sph1p bind to and are localized by the same component. The identification of a 150–amino acid domain necessary and sufficient for localization of Spa2p to sites of polarized growth and the existence of this domain in another yeast protein Sph1p suggest that the early localization of these proteins may be mediated by a receptor that recognizes this small domain.Polarized cell growth and division are essential cellular processes that play a crucial role in the development of eukaryotic organisms. Cell fate can be determined by cell asymmetry during cell division (Horvitz and Herskowitz, 1992; Cohen and Hyman, 1994; Rhyu and Knoblich, 1995). Consequently, the molecules involved in the generation and maintenance of cell asymmetry are important in the process of cell fate determination. Polarized growth can occur in response to external signals such as growth towards a nutrient (Rodriguez-Boulan and Nelson, 1989; Eaton and Simons, 1995) or hormone (Jackson and Hartwell, 1990a , b ; Segall, 1993; Keynes and Cook, 1995) and in response to internal signals as in Caenorhabditis elegans (Goldstein et al., 1993; Kimble, 1994; Priess, 1994) and Drosophila melanogaster (St Johnston and Nusslein-Volhard, 1992; Anderson, 1995) early development. Saccharomyces cerevisiae undergo polarized growth towards an external cue during mating and to an internal cue during budding. Polarization towards a mating partner (shmoo formation) and towards a new bud site requires a number of proteins (Chenevert, 1994; Chant, 1996; Drubin and Nelson, 1996). Many of these proteins are necessary for both processes and are localized to sites of polarized growth, identified by the insertion of new cell wall material (Tkacz and Lampen, 1972; Farkas et al., 1974; Lew and Reed, 1993) to the shmoo tip, bud tip, and mother–daughter bud neck. In yeast, proteins localized to growth sites include cytoskeletal proteins (Adams and Pringle, 1984; Kilmartin and Adams, 1984; Ford, S.K., and J.R. Pringle. 1986. Yeast. 2:S114; Drubin et al., 1988; Snyder, 1989; Snyder et al., 1991; Amatruda and Cooper, 1992; Lew and Reed, 1993; Waddle et al., 1996), neck filament components (septins) (Byers and Goetsch, 1976; Kim et al., 1991; Ford and Pringle, 1991; Haarer and Pringle, 1987; Longtine et al., 1996), motor proteins (Lillie and Brown, 1994), G-proteins (Ziman, 1993; Yamochi et al., 1994; Qadota et al., 1996), and two membrane proteins (Halme et al., 1996; Roemer et al., 1996; Qadota et al., 1996). Septins, actin, and actin-associated proteins localize early in the cell cycle, before a bud or shmoo tip is recognizable. How this group of proteins is localized to and maintained at sites of cell growth remains unclear.Spa2p is one of the first proteins involved in bud formation to localize to the incipient bud site before a bud is recognizable (Snyder, 1989; Snyder et al., 1991; Chant, 1996). Spa2p has been localized to where a new bud will form at approximately the same time as actin patches concentrate at this region (Snyder et al., 1991). An understanding of how Spa2p localizes to incipient bud sites will shed light on the very early stages of cell polarization. Later in the cell cycle, Spa2p is also found at the mother–daughter bud neck in cells undergoing cytokinesis. Spa2p, a nonessential protein, has been shown to be involved in bud site selection (Snyder, 1989; Zahner et al., 1996), shmoo formation (Gehrung and Snyder, 1990), and mating (Gehrung and Snyder, 1990; Chenevert et al., 1994; Yorihuzi and Ohsumi, 1994; Dorer et al., 1995). Genetic studies also suggest that Spa2p has a role in cytokinesis (Flescher et al., 1993), yet little is known about how this protein is localized to sites of polarized growth.We have used Spa2p green fluorescent protein (GFP)¹ fusions to investigate the early localization of Spa2p to sites of polarized growth in living cells. Our results demonstrate that a small domain of ∼150 amino acids of this large 1,466-residue protein is sufficient for targeting to sites of polarized growth and is necessary for Spa2p function. Furthermore, we have identified and characterized a novel yeast protein, Sph1p, which has homology to both the Spa2p amino terminus and the Spa2p localization domain. Sph1p localizes to similar regions of polarized growth and sph1 mutants have similar phenotypes as spa2 mutants.     

Dbf4-Cdc7 Phosphorylation of Mcm2 Is Required for Cell Growth

The Dbf4-Cdc7 kinase (DDK) is required for the activation of the origins of replication, and DDK phosphorylates Mcm2 in vitro. We find that budding yeast Cdc7 alone exists in solution as a weakly active multimer. Dbf4 forms a likely heterodimer with Cdc7, and this species phosphorylates Mcm2 with substantially higher specific activity. Dbf4 alone binds tightly to Mcm2, whereas Cdc7 alone binds weakly to Mcm2, suggesting that Dbf4 recruits Cdc7 to phosphorylate Mcm2. DDK phosphorylates two serine residues of Mcm2 near the N terminus of the protein, Ser-164 and Ser-170. Expression of mcm2-S170A is lethal to yeast cells that lack endogenous MCM2 (mcm2Δ); however, this lethality is rescued in cells harboring the DDK bypass mutant mcm5-bob1. We conclude that DDK phosphorylation of Mcm2 is required for cell growth.The Cdc7 protein kinase is required throughout the yeast S phase to activate origins (1, 2). The S phase cyclin-dependent kinase also activates yeast origins of replication (3–5). It has been proposed that Dbf4 activates Cdc7 kinase in S phase, and that Dbf4 interaction with Cdc7 is essential for Cdc7 kinase activity (6). However, it is not known how Dbf4-Cdc7 (DDK)² acts during S phase to trigger the initiation of DNA replication. DDK has homologs in other eukaryotic species, and the role of Cdc7 in activation of replication origins during S phase may be conserved (7–10).The Mcm2-7 complex functions with Cdc45 and GINS to unwind DNA at a replication fork (11–15). A mutation of MCM5 (mcm5-bob1) bypasses the cellular requirements for DBF4 and CDC7 (16), suggesting a critical physiologic interaction between Dbf4-Cdc7 and Mcm proteins. DDK phosphorylates Mcm2 in vitro with proteins purified from budding yeast (17, 18) or human cells (19). Furthermore, there are mutants of MCM2 that show synthetic lethality with DBF4 mutants (6, 17), suggesting a biologically relevant interaction between DBF4 and MCM2. Nevertheless, the physiologic role of DDK phosphorylation of Mcm2 is a matter of dispute. In human cells, replacement of MCM2 DDK-phosphoacceptor residues with alanines inhibits DNA replication, suggesting that Dbf4-Cdc7 phosphorylation of Mcm2 in humans is important for DNA replication (20). In contrast, mutation of putative DDK phosphorylation sites at the N terminus of Schizosaccharomyces pombe Mcm2 results in viable cells, suggesting that phosphorylation of S. pombe Mcm2 by DDK is not critical for cell growth (10).In budding yeast, Cdc7 is present at high levels in G₁ and S phase, whereas Dbf4 levels peak in S phase (18, 21, 22). Furthermore, budding yeast DDK binds to chromatin during S phase (6), and it has been shown that Dbf4 is required for Cdc7 binding to chromatin in budding yeast (23, 24), fission yeast (25), and Xenopus (9). Human and fission yeast Cdc7 are inert on their own (7, 8), but Dbf4-Cdc7 is active in phosphorylating Mcm proteins in budding yeast (6, 26), fission yeast (7), and human (8, 10). Based on these data, it has been proposed that Dbf4 activates Cdc7 kinase in S phase and that Dbf4 interaction with Cdc7 is essential for Cdc7 kinase activity (6, 9, 18, 21–24). However, a mechanistic analysis of how Dbf4 activates Cdc7 has not yet been accomplished. For example, the multimeric state of the active Dbf4-Cdc7 complex is currently disputed. A heterodimer of fission yeast Cdc7 (Hsk1) in complex with fission yeast Dbf4 (Dfp1) can phosphorylate Mcm2 (7). However, in budding yeast, oligomers of Cdc7 exist in the cell (27), and Dbf4-Cdc7 exists as oligomers of 180 and 300 kDa (27).DDK phosphorylates the N termini of human Mcm2 (19, 20, 28), human Mcm4 (10), budding yeast Mcm4 (26), and fission yeast Mcm6 (10). Although the sequences of the Mcm N termini are poorly conserved, the DDK sites identified in each study have neighboring acidic residues. The residues of budding yeast Mcm2 that are phosphorylated by DDK have not yet been identified.In this study, we find that budding yeast Cdc7 is weakly active as a multimer in phosphorylating Mcm2. However, a low molecular weight form of Dbf4-Cdc7, likely a heterodimer, has a higher specific activity for phosphorylation of Mcm2. Dbf4 or DDK, but not Cdc7, binds tightly to Mcm2, suggesting that Dbf4 recruits Cdc7 to Mcm2. DDK phosphorylates two serine residues of Mcm2, Ser-164 and Ser-170, in an acidic region of the protein. Mutation of Ser-170 is lethal to yeast cells, but this phenotype is rescued by the DDK bypass mutant mcm5-bob1. We conclude that DDK phosphorylation of Ser-170 of Mcm2 is required for budding yeast growth.     

Tyrosine Phosphorylation of Growth Factor Receptor-bound Protein-7 by Focal Adhesion Kinase in the Regulation of Cell Migration, Proliferation, and Tumorigenesis

We have previously reported that growth factor receptor-bound protein-7 (Grb7), an Src-homology 2 (SH2)-containing adaptor protein, enables interaction with focal adhesion kinase (FAK) to regulate cell migration in response to integrin activation. To further elucidate the signaling events mediated by FAK·Grb7 complexes in promoting cell migration and other cellular functions, we firstly examined the phos pho ryl a ted tyrosine site(s) of Grb7 by FAK using an in vivo mutagenesis. We found that FAK was capable of phos pho rylating at least 2 of 12 tyrosine residues within Grb7, Tyr-188 and Tyr-338. Moreover, mutations converting the identified Tyr to Phe inhibited integrin-dependent cell migration as well as impaired cell proliferation but not survival compared with the wild-type control. Interestingly, the above inhibitory effects caused by the tyrosine phos pho ryl a tion-deficient mutants are probably attributed to their down-regulation of phospho-Tyr-397 of FAK, thereby implying a mechanism by competing with wild-type Grb7 for binding to FAK. Consequently, these tyrosine phos pho ryl a tion-deficient mutants evidently altered the phospho-Tyr-118 of paxillin and phos pho ryl a tion of ERK1/2 but less on phospho-Ser-473 of AKT, implying their involvement in the FAK·Grb7-mediated cellular functions. Additionally, we also illustrated that the formation of FAK·Grb7 complexes and Grb7 phos pho ryl a tion by FAK in an integrin-dependent manner were essential for cell migration, proliferation and anchorage-independent growth in A431 epidermal carcinoma cells, indicating the importance of FAK·Grb7 complexes in tumorigenesis. Our data provide a better understanding on the signal transduction event for FAK·Grb7-mediated cellular functions as well as to shed light on a potential therapeutic in cancers.Growth factor receptor bound protein-7 (Grb7)² is initially identified as a SH2 domain-containing adaptor protein bound to the activated EGF receptor (1). Grb7 is composed of an N-terminal proline-rich region, following a putative RA (Ras-associating) domain and a central PH (pleckstrin homology) domain and a BPS motif (between PH and SH2 domains), and a C-terminal SH2 domain (2–6). Despite the lack of enzymatic activity, the presence of multiple protein-protein interaction domains allows Grb7 family adaptor proteins to participate in versatile signal transduction pathways and, therefore, to regulate many cellular functions (4–6). A number of signaling molecules has been reported to interact with these featured domains, although most of the identified Grb7 binding partners are mediated through its SH2 domain. For example, the SH2 domain of Grb7 has been demonstrated to be capable of binding to the phospho-tyrosine sites of EGF receptor (1), ErbB2 (7), ErbB3 and ErbB4 (8), Ret (9), platelet-derived growth factor receptor (10), insulin receptor (11), SHPTP2 (12), Tek/Tie2 (13), caveolin (14), c-Kit (15), EphB1 (16), G6f immunoreceptor protein (17), Rnd1 (18), Shc (7), FAK (19), and so on. The proceeding α-helix of the PH domain of Grb7 is the calmodulin-binding domain responsible for recruiting Grb7 to plasma membrane in a Ca²⁺-dependent manner (20), and the association between the PH domain of Grb7 and phosphoinositides is required for the phosphorylation by FAK (21). Two additional proteins, NIK (nuclear factor κB-inducing kinase) and FHL2 (four and half lim domains isoform 2), in association with the GM region (Grb and Mig homology region) of Grb7 are also reported, although the physiological functions for these interactions remain unknown (22, 23). Recently, other novel roles in translational controls and stress responses through the N terminus of Grb7 are implicated for the findings of Grb7 interacting with the 5′-untranslated region of capped targeted KOR (kappa opioid receptor) mRNA and the Hu antigen R of stress granules in an FAK-mediated phosphorylation manner (24, 25).Unlike its member proteins Grb10 and Grb14, the role of Grb7 in cell migration is unambiguous and well documented. This is supported by a series of studies. Firstly, Grb7 family members share a significantly conserved molecular architecture with the Caenorhabditis elegans Mig-10 protein, which is involved in neuronal cell migration during embryonic development (4, 5, 26), suggesting that Grb7 may play a role in cell migration. Moreover, Grb7 is often co-amplified with Her2/ErbB2 in certain human cancers and tumor cell lines (7, 27, 28), and its overexpression resulted in invasive and metastatic consequences of various cancers and tumor cells (23, 29–33). On the contrary, knocking down Grb7 by RNA interference conferred to an inhibitory outcome of the breast cancer motility (34). Furthermore, interaction of Grb7 with autophosphorylated FAK at Tyr-397 could promote integrin-mediated cell migration in NIH 3T3 and CHO cells, whereas overexpression of its SH2 domain, an dominant negative mutant of Grb7, inhibited cell migration (19, 35). Recruitment and phosphorylation of Grb7 by EphB1 receptors enhanced cell migration in an ephrin-dependent manner (16). Recently, G7–18NATE, a selective Grb7-SH2 domain affinity cyclic peptide, was demonstrated to efficiently block cell migration of tumor cells (32, 36). In addition to cell migration, Grb7 has been shown to play a role in a variety of physiological and pathological events, for instance, kidney development (37), tumorigenesis (7, 14, 38–41), angiogenic activity (20), proliferation (34, 42, 43), anti-apoptosis (44), gene expression regulation (24), Silver-Russell syndrome (45), rheumatoid arthritis (46), atopic dermatitis (47), and T-cell activation (17, 48). Nevertheless, it remains largely unknown regarding the downstream signaling events of Grb7-mediated various functions. In particular, given the role of Grb7 as an adaptor molecule and its SH2 domain mainly interacting with upstream regulators, it will be interesting to identify potential downstream effectors through interacting with the functional GM region or N-terminal proline-rich region.In this report, we identified two tyrosine phosphorylated sites of Grb7 by FAK and deciphered the signaling targets downstream through these phosphorylated tyrosine sites to regulate various cellular functions such as cell migration, proliferation, and survival. In addition, our study sheds light on tyrosine phosphorylation of Grb7 by FAK involved in tumorigenesis.     

Deep Proteomic Evaluation of Primary and Cell Line Motoneuron Disease Models Delineates Major Differences in Neuronal Characteristics

The fatal neurodegenerative disorders amyotrophic lateral sclerosis and spinal muscular atrophy are, respectively, the most common motoneuron disease and genetic cause of infant death. Various in vitro model systems have been established to investigate motoneuron disease mechanisms, in particular immortalized cell lines and primary neurons. Using quantitative mass-spectrometry-based proteomics, we compared the proteomes of primary motoneurons to motoneuron-like cell lines NSC-34 and N2a, as well as to non-neuronal control cells, at a depth of 10,000 proteins. We used this resource to evaluate the suitability of murine in vitro model systems for cell biological and biochemical analysis of motoneuron disease mechanisms. Individual protein and pathway analysis indicated substantial differences between motoneuron-like cell lines and primary motoneurons, especially for proteins involved in differentiation, cytoskeleton, and receptor signaling, whereas common metabolic pathways were more similar. The proteins associated with amyotrophic lateral sclerosis also showed distinct differences between cell lines and primary motoneurons, providing a molecular basis for understanding fundamental alterations between cell lines and neurons with respect to neuronal pathways with relevance for disease mechanisms. Our study provides a proteomics resource for motoneuron research and presents a paradigm of how mass-spectrometry-based proteomics can be used to evaluate disease model systems.Motoneurons are extremely extended neurons that mediate the control of all muscle types by the central nervous system. Therefore, diseases involving progressive motoneuron degeneration such as amyotrophic lateral sclerosis (ALS)¹ (OMIM: 105400) or spinal muscle atrophy (OMIM: 253300) are particularly devastating and generally fatal disorders. Today, ALS is believed to form a phenotypic continuum with the disease entity frontotemporal lobe degeneration (OMIM: 600274) (1, 2). About 10% of ALS cases are known to be inherited, but the vast majority are considered sporadic. The number of inherited cases might be underestimated because of incomplete family histories, non-paternity, early death of family members, or incomplete penetrance (3).Mutations in several genes have been reported for the familial form, including in Sod1 (4), Als2 (5), Setx (6), Vapb (7), Tardbp (8, 9), Fus/Tls (10, 11), Vcp (12), Pfn1 (13), and several others (reviewed in Ref. 14). The most frequent genetic cause of inherited ALS was recently shown to be a hexanucleotide repeat expansion in an intron of a gene of unknown function called C9orf72 (15–17). Based on the spectrum of known mutations, several disease mechanisms for ALS have been proposed, including dysfunction of protein folding, axonal transport, RNA splicing, and metabolism (reviewed in Refs. 14, 18, and 19). Despite intensive research, it is still unclear whether a main common molecular pathway or mechanism underlies motoneuron degeneration in ALS and frontotemporal lobe degeneration. Spinal muscle atrophy is caused by homozygous mutations or deletions in the survival of motor neuron gene (Smn1) that presumably impair the RNA metabolism through diminished functionality of the Smn1 gene product (20). Over recent decades several model systems have been established to investigate ALS (21). These include transgenic animal models such as mouse (22), drosophila (23), and zebrafish (24). In cell-based studies, primary motoneurons cultured from rodent embryos (25) or motoneuron-like cell lines are employed. Primary cells are considered to more closely mimic the in vivo situation, but they are more challenging to establish and maintain. In contrast, the degree of functional relevance of cell lines can be difficult to establish, but they can be propagated without limitation and are well suited for high-throughput analysis. In particular, the spinal cord neuron–neuroblastoma hybrid cell line NSC-34 (26) and the mouse neuroblastoma cell line N2a (27) are widely used not only to assess motoneuron function, but also to study disease mechanisms in motoneurons (28, 29).As proteins are the functional actors in cells, proteomics should be able to make important contributions to the characterization and evaluation of cellular models. In particular, by identifying and quantifying the expressed proteins and bioinformatically interpreting the results, one can obtain enough information to infer functional differences. Our laboratory has previously shown proof of concept of such an approach by comparing the expression levels of about 4,000 proteins between primary hepatocytes and a hepatoma cell line (30). Very recently, mass-spectrometry-based proteomics has achieved sufficient depth and accuracy to quantify almost the entire proteome of mammalian cell lines (31–33). Furthermore, new instrumentation and algorithms now make it possible to perform label-free quantification between multiple cellular systems and with an accuracy previously associated only with stable isotope labeling techniques (34, 35).To evaluate the suitability of motoneuron-like cell lines as cellular model systems for research on ALS and related disorders, we characterized the proteomes of two widely used cell lines, NSC-34 and N2a, and compared them with the proteomes of mouse primary motoneurons and non-neuronal control cell lines. To generate primary motoneurons, we employed a recently described culturing system that makes it possible to isolate highly enriched motoneuron populations in less than 8 h (25). We identified more than 10,000 proteins and investigated differences in quantitative levels of individual neuron-associated proteins and pathways related to motoneuron function and disease mechanisms.     

Succinylome Analysis Reveals the Involvement of Lysine Succinylation in Metabolism in Pathogenic Mycobacterium tuberculosis

Mycobacterium tuberculosis (Mtb), the causative agent of human tuberculosis, remains one of the most prevalent human pathogens and a major cause of mortality worldwide. Metabolic network is a central mediator and defining feature of the pathogenicity of Mtb. Increasing evidence suggests that lysine succinylation dynamically regulates enzymes in carbon metabolism in both bacteria and human cells; however, its extent and function in Mtb remain unexplored. Here, we performed a global succinylome analysis of the virulent Mtb strain H37Rv by using high accuracy nano-LC-MS/MS in combination with the enrichment of succinylated peptides from digested cell lysates and subsequent peptide identification. In total, 1545 lysine succinylation sites on 626 proteins were identified in this pathogen. The identified succinylated proteins are involved in various biological processes and a large proportion of the succinylation sites are present on proteins in the central metabolism pathway. Site-specific mutations showed that succinylation is a negative regulatory modification on the enzymatic activity of acetyl-CoA synthetase. Molecular dynamics simulations demonstrated that succinylation affects the conformational stability of acetyl-CoA synthetase, which is critical for its enzymatic activity. Further functional studies showed that CobB, a sirtuin-like deacetylase in Mtb, functions as a desuccinylase of acetyl-CoA synthetase in in vitro assays. Together, our findings reveal widespread roles for lysine succinylation in regulating metabolism and diverse processes in Mtb. Our data provide a rich resource for functional analyses of lysine succinylation and facilitate the dissection of metabolic networks in this life-threatening pathogen.Post-translational modifications (PTMs)¹ are complex and fundamental mechanisms modulating diverse protein properties and functions, and have been associated with almost all known cellular pathways and disease processes (1, 2). Among the hundreds of different PTMs, acylations at lysine residues, such as acetylation (3–6), malonylation (7, 8), crotonylation (9, 10), propionylation (11–13), butyrylation (11, 13), and succinylation (7, 14–16) are crucial for functional regulations of many prokaryotic and eukaryotic proteins. Because these lysine PTMs depend on the acyl-CoA metabolic intermediates, such as acetyl-CoA (Ac-CoA), succinyl-CoA, and malonyl-CoA, lysine acylation could provide a mechanism to respond to changes in the energy status of the cell and regulate energy metabolism and the key metabolic pathways in diverse organisms (17, 18).Among these lysine PTMs, lysine succinylation is a highly dynamic and regulated PTM defined as transfer of a succinyl group (-CO-CH₂-CH₂-CO-) to a lysine residue of a protein molecule (8). It was recently identified and comprehensively validated in both bacterial and mammalian cells (8, 14, 16). It was also identified in core histones, suggesting that lysine succinylation may regulate the functions of histones and affect chromatin structure and gene expression (7). Accumulating evidence suggests that lysine succinylation is a widespread and important PTM in both eukaryotes and prokaryotes and regulates diverse cellular processes (16). The system-wide studies involving lysine-succinylated peptide immunoprecipitation and liquid chromatography-mass spectrometry (LC-MS/MS) have been employed to analyze the bacteria (E. coli) (14, 16), yeast (S. cerevisiae), human (HeLa) cells, and mouse embryonic fibroblasts and liver cells (16, 19). These succinylome studies have generated large data sets of lysine-succinylated proteins in both eukaryotes and prokaryotes and demonstrated the diverse cellular functions of this PTM. Notably, lysine succinylation is widespread among diverse mitochondrial metabolic enzymes that are involved in fatty acid metabolism, amino acid degradation, and the tricarboxylic acid cycle (19, 20). Thus, lysine succinylation is reported as a functional PTM with the potential to impact mitochondrial metabolism and coordinate different metabolic pathways in human cells and bacteria (14, 19–22).Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), is a major cause of mortality worldwide and claims more human lives annually than any other bacterial pathogen (23). About one third of the world''s population is infected with Mtb, which leads to nearly 1.3 million deaths and 8.6 million new cases of TB in 2012 worldwide (24). Mtb remains a major threat to global health, especially in the developing countries. Emergence of multidrug resistant (MDR) and extensively drug-resistant (XDR) Mtb, and also the emergence of co-infection between TB and HIV have further worsened the situation (25–27). Among bacterial pathogens, Mtb has a distinctive life cycle spanning different environments and developmental stages (28). Especially, Mtb can exist in dormant or active states in the host, leading to asymptomatic latent TB infection or active TB disease (29). To achieve these different physiologic states, Mtb developed a mechanism to sense diverse signals from the host and to coordinately regulate multiple cellular processes and pathways (30, 31). Mtb has evolved its metabolic network to both maintain and propagate its survival as a species within humans (32–35). It is well accepted that metabolic network is a central mediator and defining feature of the pathogenicity of Mtb (23, 36–38). Knowledge of the regulation of metabolic pathways used by Mtb during infection is therefore important for understanding its pathogenicity, and can also guide the development of novel drug therapies (39). On the other hand, increasing evidence suggests that lysine succinylation dynamically regulates enzymes in carbon metabolism in both bacteria and human cells (14, 19–22). It is tempting to speculate that lysine succinylation may play an important regulatory role in metabolic processes in Mtb. However, to the best of our knowledge, no succinylated protein in Mtb has been identified, presenting a major obstacle to understand the regulatory roles of lysine succinylation in this life-threatening pathogen.In order to fill this gap in our knowledge, we have initiated a systematic study of the identities and functional roles of the succinylated protein in Mtb. Because Mtb H37Rv is the first sequenced Mtb strain (40) and has been extensively used for studies in dissecting the roles of individual genes in pathogenesis (41), it was selected as a test case. We analyzed the succinylome of Mtb H37Rv by using high accuracy nano-LC-MS/MS in combination with the enrichment of succinylated peptides from digested cell lysates and subsequent peptide identification. In total, 1545 lysine succinylation sites on 626 proteins were identified in this pathogen. The identified succinylated proteins are involved in various biological processes and render particular enrichment to metabolic process. A large proportion of the succinylation sites are present on proteins in the central metabolism pathway. We further dissected the regulatory role of succinylation on acetyl-CoA synthetase (Acs) via site-specific mutagenesis analysis and molecular dynamics (MD) simulations showed that reversible lysine succinylation could inhibit the activity of Acs. Further functional studies showed that CobB, a sirtuin-like deacetylase in Mtb, functions as a deacetylase and as a desuccinylase of Acs in in vitro assays. Together, our findings provide significant insights into the range of functions regulated by lysine succinylation in Mtb.     

Hypomaturation Enamel Defects in Klk4 Knockout/LacZ Knockin Mice

Kallikrein 4 (Klk4) is believed to play an essential role in enamel biomineralization, because defects in KLK4 cause hypomaturation amelogenesis imperfecta. We used gene targeting to generate a knockin mouse that replaces the Klk4 gene sequence, starting at the translation initiation site, with a lacZ reporter gene. Correct targeting of the transgene was confirmed by Southern blot and PCR analyses. Histochemical X-gal (5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside) staining demonstrated expression of β-galactosidase in maturation stage ameloblasts. No X-gal staining was observed in secretory stage ameloblasts or in odontoblasts. Retained enamel proteins were observed in the maturation stage enamel of the Klk4 null mouse, but not in the Klk4 heterozygous or wild-type mice. The enamel layer in the Klk4 null mouse was normal in thickness and contained decussating enamel rods but was rapidly abraded following weaning, despite the mice being maintained on soft chow. In function the enamel readily fractured within the initial rod and interrod enamel above the parallel enamel covering the dentino-enamel junction. Despite the lack of Klk4 and the retention of enamel proteins, significant levels of crystal maturation occurred (although delayed), and the enamel achieved a mineral density in some places greater than that detected in bone and dentin. An important finding was that individual enamel crystallites of erupted teeth failed to grow together, interlock, and function as a unit. Instead, individual crystallites seemed to spill out of the enamel when fractured. These results demonstrate that Klk4 is essential for the removal of enamel proteins and the proper maturation of enamel crystals.Dental enamel is composed of highly ordered, very long crystals of calcium hydroxyapatite (Ca₁₀(PO₄)₆(OH)₂). Mature enamel crystallites are about 70 nm wide and 30 nm thick, but are of unmeasurable length (1), probably extending all the way from the dentin layer to the surface of the tooth (2). Enamel crystallites are organized into bundles called rods, with about 10,000 parallel crystals in a rod (3). Each enamel rod is the product of a single ameloblast, the cell type that forms a continuous sheet over the developing enamel and orchestrates its formation. Dental enamel of erupted teeth is ∼95% mineral (by weight) (4), with most of the non-mineral component being water. Protein comprises <1% of its weight. Forming enamel, however, is over 30% protein (5). Much of the protein is reabsorbed by ameloblasts and degraded in lysosomes (6, 7), but extracellular proteases also play a role in matrix protein removal (8–10).Dental enamel formation is divided into secretory, transition, and maturation stages (11, 12). During the secretory stage, enamel crystals grow primarily in length. As the crystals extend, the enamel layer expands. Enamel crystallites lengthen along a mineralization front at the secretory surface of the ameloblast cell membrane. There, mineral deposits rapidly on the crystallite tips, and very slowly on their sides (3, 13, 14). By the end of the secretory stage the enamel crystals are full-length and the enamel layer as a whole is as thick as it will ever be, but it has only about 14% of the mineral as it will have when the tooth erupts (15). Following the secretory stage there is then a transition during which the ameloblasts greatly reduce their secretion of enamel proteins (16) and convert to maturation ameloblasts (17). During the maturation stage, mineral is deposited exclusively on the sides of pre-existing enamel crystallites (18), which grow in width and thickness until further growth is prevented by contact with adjacent crystals (19, 20). During early maturation the percentage protein by weight drops from 30 to 2% (5), and half of the total enamel mineral is deposited. The final 30–35% of mineral is deposited in the absence of significant protein and allows the crystals to grow firmly against one another and to mechanically interlock (15).The major secretory stage enamel proteins are amelogenin (21, 22), ameloblastin (23–25), and enamelin (26, 27). These proteins function specifically during enamel formation, and the disease phenotypes exhibited by mice lacking these genes are confined to the developing teeth and include enamel agenesis (28–30). These genes are often deleted or are reduced to pseudogenes in vertebrates such as birds or baleen whales that evolved alternatives to developing teeth (31, 32). Although the enamel extracellular matrix proteins are critical for growing enamel crystals, they are not part of the final enamel product. Prior to tooth eruption, enamel proteins are digested by proteases and reabsorbed by ameloblasts. Two extracellular matrix proteases are involved in the cleavage of enamel proteins: matrix metalloproteinase 20 (Mmp-20)² (33) and kallikrein 4 (Klk4) (34).Mmp-20 is secreted along with amelogenin, ameloblastin, and enamelin by secretory stage ameloblasts (35–37). Mmp-20 activity can account for the range of cleavages observed in secretory stage enamel proteins (38) and appears to be the only protease secreted by ameloblasts during the secretory stage. Mmp20-null mice have enamel that is thinner and softer than normal, lacks enamel rod organization, and tends to chip off the crown surface (39, 40). Like the other secretory stage enamel proteins, Mmp20 expression appears to be restricted to developing teeth (41), as is the diseased phenotype when the human gene is defective (42–44).Klk4 is a serine protease that is secreted by transition and maturation stage ameloblasts but is not expressed by secretory stage ameloblasts (45, 46). Klk4 might also be expressed by odontoblasts, the cells that form dentin (47). Klk4 has broad substrate specificity (48, 49) and is capable of activating other proteases (50–52) and protease activated receptors (53, 54). Unlike most proteins secreted by ameloblasts, Klk4 is expressed in other tissues, most notably the prostate (55) and endometrium (56). Much attention has been focused on the potential role of Klk4 in cancers. Klk4 is increased in breast cancer stromal cells (57), in prostate cancer cells (58–61), and ovarian cancer cells (62–65). Despite this focus on the potential role of Klk4 in tumors, very little is known about the normal expression and function of Klk4 in nondental tissues. A loss of function mutation in both human KLK4 alleles caused a hypomaturation enamel phenotype in the absence of any observable defects elsewhere in the body (66).To gain insights into the role of Klk4 in normal dental enamel formation, and to better characterize the normal temporal and spatial patterns of Klk4 expression, we have used gene targeting to knock out normal Klk4 expression, while replacing the Klk4 code with lacZ, the bacterial gene encoding β-galactosidase reporter in mice. We demonstrate that Klk4 is not expressed by secretory stage ameloblasts, but is specifically expressed by ameloblasts later in enamel formation and is necessary for the proper removal of enamel proteins, the final thickening of enamel crystals, and ultimately, for hardening of the enamel layer.     

N-Terminal-oriented Proteogenomics of the Marine Bacterium Roseobacter Denitrificans Och114 using N-Succinimidyloxycarbonylmethyl)tris(2,4,6-trimethoxyphenyl)phosphonium bromide (TMPP) Labeling and Diagonal Chromatography

Given the ease of whole genome sequencing with next-generation sequencers, structural and functional gene annotation is now purely based on automated prediction. However, errors in gene structure are frequent, the correct determination of start codons being one of the main concerns. Here, we combine protein N termini derivatization using (N-Succinimidyloxycarbonylmethyl)tris(2,4,6-trimethoxyphenyl)phosphonium bromide (TMPP Ac-OSu) as a labeling reagent with the COmbined FRActional DIagonal Chromatography (COFRADIC) sorting method to enrich labeled N-terminal peptides for mass spectrometry detection. Protein digestion was performed in parallel with three proteases to obtain a reliable automatic validation of protein N termini. The analysis of these N-terminal enriched fractions by high-resolution tandem mass spectrometry allowed the annotation refinement of 534 proteins of the model marine bacterium Roseobacter denitrificans OCh114. This study is especially efficient regarding mass spectrometry analytical time. From the 534 validated N termini, 480 confirmed existing gene annotations, 41 highlighted erroneous start codon annotations, five revealed totally new mis-annotated genes; the mass spectrometry data also suggested the existence of multiple start sites for eight different genes, a result that challenges the current view of protein translation initiation. Finally, we identified several proteins for which classical genome homology-driven annotation was inconsistent, questioning the validity of automatic annotation pipelines and emphasizing the need for complementary proteomic data. All data have been deposited to the ProteomeXchange with identifier PXD000337.Recent developments in mass spectrometry and bioinformatics have established proteomics as a common and powerful technique for identifying and quantifying proteins at a very broad scale, but also for characterizing their post-translational modifications and interaction networks (1, 2). In addition to the avalanche of proteomic data currently being reported, many genome sequences are established using next-generation sequencing, fostering proteomic investigations of new cellular models. Proteogenomics is a relatively recent field in which high-throughput proteomic data is used to verify coding regions within model genomes to refine the annotation of their sequences (2–8). Because genome annotation is now fully automated, the need for accurate annotation for model organisms with experimental data is crucial. Many projects related to genome re-annotation of microorganisms with the help of proteomics have been recently reported, such as for Mycoplasma pneumoniae (9), Rhodopseudomonas palustris (10), Shewanella oneidensis (11), Thermococcus gammatolerans (12), Deinococcus deserti (13), Salmonella thyphimurium (14), Mycobacterium tuberculosis (15, 16), Shigella flexneri (17), Ruegeria pomeroyi (18), and Candida glabrata (19), as well as for higher organisms such as Anopheles gambiae (20) and Arabidopsis thaliana (4, 5).The most frequently reported problem in automatic annotation systems is the correct identification of the translational start codon (21–23). The error rate depends on the primary annotation system, but also on the organism, as reported for Halobacterium salinarum and Natromonas pharaonis (24), Deinococcus deserti (21), and Ruegeria pomeroyi (18), where the error rate is estimated above 10%. Identification of a correct translational start site is essential for the genetic and biochemical analysis of a protein because errors can seriously impact subsequent biological studies. If the N terminus is not correctly identified, the protein will be considered in either a truncated or extended form, leading to errors in bioinformatic analyses (e.g. during the prediction of its molecular weight, isoelectric point, cellular localization) and major difficulties during its experimental characterization. For example, a truncated protein may be heterologously produced as an unfolded polypeptide recalcitrant to structure determination (25). Moreover, N-terminal modifications, which are poorly documented in annotation databases, may occur (26, 27).Unfortunately, the poor polypeptide sequence coverage obtained for the numerous low abundance proteins in current shotgun MS/MS proteomic studies implies that the overall detection of N-terminal peptides obtained in proteogenomic studies is relatively low. Different methods for establishing the most extensive list of protein N termini, grouped under the so-called “N-terminomics” theme, have been proposed to selectively enrich or improve the detection of these peptides (2, 28, 29). Large N-terminome studies have recently been reported based on resin-assisted enrichment of N-terminal peptides (30) or terminal amine isotopic labeling of substrates (TAILS) coupled to depletion of internal peptides with a water-soluble aldehyde-functionalized polymer (31–35). Among the numerous N-terminal-oriented methods (2), specific labeling of the N terminus of intact proteins with N-tris(2,4,6-trimethoxyphenyl)phosphonium acetyl succinamide (TMPP-Ac-OSu)¹ has proven reliable (21, 36–39). TMPP-derivatized N-terminal peptides have interesting properties for further LC-MS/MS mass spectrometry: (1) an increase in hydrophobicity because of the trimethoxyphenyl moiety added to the peptides, increasing their retention times in reverse phase chromatography, (2) improvement of their ionization because of the introduction of a positively charged group, and (3) a much simpler fragmentation pattern in tandem mass spectrometry. Other reported approaches rely on acetylation, followed by trypsin digestion, and then biotinylation of free amino groups (40); guanidination of lysine lateral chains followed by N-biotinylation of the N termini and trypsin digestion (41); or reductive amination of all free amino groups with formaldehyde preceeding trypsin digestion (42). Recently, we applied the TMPP method to the proteome of the Deinococcus deserti bacterium isolated from upper sand layers of the Sahara desert (13). This method enabled the detection of N-terminal peptides allowing the confirmation of 278 translation initiation codons, the correction of 73 translation starts, and the identification of non-canonical translation initiation codons (21). However, most TMPP-labeled N-terminal peptides are hidden among the more abundant internal peptides generated after proteolysis of a complex proteome, precluding their detection. This results in disproportionately fewer N-terminal validations, that is, 5 and 8% of total polypeptides coded in the theoretical proteomes of Mycobacterium smegmatis (37) and Deinococcus deserti (21) with a total of 342 and 278 validations, respectively.An interesting chromatographic method to fractionate peptide mixtures for gel-free high-throughput proteome analysis has been developed over the last years and applied to various topics (43, 44). This technique, known as COmbined FRActional DIagonal Chromatography (COFRADIC), uses a double chromatographic separation with a chemical reaction in between to change the physico-chemical properties of the extraneous peptides to be resolved from the peptides of interest. Its previous applications include the separation of methionine-containing peptides (43), N-terminal peptide enrichment (45, 46), sulfur amino acid-containing peptides (47), and phosphorylated peptides (48). COFRADIC was identified as the best method for identification of N-terminal peptides of two archaea, resulting in the identification of 240 polypeptides (9% of the theoretical proteome) for Halobacterium salinarum and 220 (8%) for Natronomonas pharaonis (24).Taking advantage of both the specificity of TMPP labeling, the resolving power of COFRADIC for enrichment, and the increase in information through the use of multiple proteases, we performed the proteogenomic analysis of a marine bacterium from the Roseobacter clade, namely Roseobacter denitrificans OCh114. This novel approach allowed us to validate and correct 534 unique proteins (13% of the theoretical proteome) with TMPP-labeled N-terminal signatures obtained using high-resolution tandem mass spectrometry. We corrected 41 annotations and detected five new open reading frames in the R. denitrificans genome. We further identified eight distinct proteins showing direct evidence for multiple start sites.     

Laserspray Ionization, a New Atmospheric Pressure MALDI Method for Producing Highly Charged Gas-phase Ions of Peptides and Proteins Directly from Solid Solutions

The first example of a matrix-assisted laser desorption/ionization (MALDI) process producing multiply charged mass spectra nearly identical to those observed with electrospray ionization (ESI) is presented. MALDI is noted for its ability to produce singly charged ions, but in the experiments described here multiply charged ions are produced by laser ablation of analyte incorporated into a common MALDI matrix, 2,5-dihydroxybenzoic acid, using standard solvent-based sample preparation protocols. Laser ablation is known to produce matrix clusters in MALDI provided a threshold energy is achieved. We propose that these clusters (liquid droplets) are highly charged, and under conditions that produce sufficient matrix evaporation, ions are field-evaporated from the droplets similarly to ESI. Because of the multiple charging, advanced mass spectrometers with limited mass-to-charge range can be used for protein characterization. Thus, using an Orbitrap mass spectrometer, low femtomole quantities of proteins produce full-range mass spectra at 100,000 mass resolution with <5-ppm mass accuracy and with 1-s acquisition. Furthermore, the first example of protein fragmentation using electron transfer dissociation with MALDI is presented.Two primary differences between ESI and MALDI methods are the sample environment (solution versus solid) and the observable charge state(s) (multiply versus singly charged). The multiply charged ions observed in ESI mass spectrometry (MS) enhance the yields of fragment ions, a key benefit in structure characterization, and allow analysis of high molecular weight compounds on mass spectrometers with a limited mass-to-charge (m/z) range. In contrast, MALDI MS is ideal for the analysis of heterogeneous samples because it often requires less sample, and spectra of singly charged ions are easier to interpret. We report here the astonishing observation of highly charged molecular ions by laser ablation of a solid matrix/analyte mixture typically used in MALDI MS analyses. The distribution and abundances of the observed ions are similar to those obtained by ESI. Importantly, the MALDI mechanism that produces singly charged ions can be “turned on” at the operator''s will by changing only the matrix or matrix preparation conditions; this capability is not available with any other ionization method. These findings show for the first time that singly charged ions as well as multiply charged ions are available in MALDI. Besides having important mechanistic implications relating to MALDI and ESI, our findings have enormous practical analytical utility.ESI and MALDI combined with MS revolutionized the study of biological materials and earned the Nobel Prize in Chemistry for their ability to ionize proteins for analysis using MS. However, after two decades of extensive studies, the mechanism for ion formation in MALDI remains controversial (1–8). At the heart of these debates lies the predominance of singly charged ions in MALDI mass spectra; the exception being very high mass compounds. A mechanism for the formation of multiply charged ions in MALDI has previously been proposed (1) based on molecular modeling studies (9, 10) and glimpses of multiply charged ions have been observed in lower molecular weight compounds (11–14). The formation of these multiply charged ions has been attributed to sample preparation, high laser fluence, a metal-free sample stage, use of an IR laser, and atmospheric pressure (AP)1 conditions. Multiply charged ions were also recently observed by laser ablation of a liquid surface in the presence of a high electric field (15). The inability in that experiment to observe ions from a solid MALDI matrix/analyte sample or in the absence of an electric field suggests an ionization process involving liquid droplets in a high field similar to ESI (16) or other liquid based, field-induced ionization methods (17, 18).Here, we show analytically useful ESI-like MALDI mass spectra obtained using standard MALDI conditions but using a nontraditional source (19) mounted in place of the standard atmospheric pressure ionization source on a mass spectrometer most commonly used with ESI. The utility of this MALDI MS method for extending the mass range of mass spectrometers as well as the capability of peptide/protein sequencing using electron transfer dissociation (ETD) (20) is demonstrated. Because highly charged ions have not previously been observed with any MALDI ion source configuration, we briefly discuss the fundamental concepts that lead to their production. Key aspects of laserspray ionization (LSI) are laser ablation using a UV laser aligned in transmission geometry (TG) (21–23), field-free (FF) at AP (24), using a heated AP to vacuum ion transfer capillary. In order to emphasize the MALDI sample preparation but distinguish laserspray from conventional AP-MALDI, the new ionization method will hereafter be referred to as FF-TG AP-MALDI.     

Mcl-1 Degradation during Hepatocyte Lipoapoptosis

The mechanisms of free fatty acid-induced lipoapoptosis are incompletely understood. Here we demonstrate that Mcl-1, an anti-apoptotic member of the Bcl-2 family, was rapidly degraded in hepatocytes in response to palmitate and stearate by a proteasome-dependent pathway. Overexpression of a ubiquitin-resistant Mcl-1 mutant in Huh-7 cells attenuated palmitate-mediated Mcl-1 loss and lipoapoptosis; conversely, short hairpin RNA-targeted knockdown of Mcl-1 sensitized these cells to lipoapoptosis. Palmitate-induced Mcl-1 degradation was attenuated by the novel protein kinase C (PKC) inhibitor rottlerin. Of the two human novel PKC isozymes, PKCδ and PKCθ, only activation of PKCθ was observed by phospho-immunoblot analysis. As compared with Jurkat cells, a smaller PKCθ polypeptide and mRNA were expressed in hepatocytes consistent with an alternative splice variant. Short hairpin RNA-mediated knockdown of PKCθ reduced Mcl-1 degradation and lipoapoptosis. Likewise, genetic deletion of Pkcθ also attenuated Mcl-1 degradation and cytotoxicity by palmitate in primary hepatocytes. During treatment with palmitate, rottlerin inhibited phosphorylation of Mcl-1 at Ser¹⁵⁹, a phosphorylation site previously implicated in Mcl-1 turnover. Consistent with these results, an Mcl-1 S159A mutant was resistant to degradation and improved cell survival during palmitate treatment. Collectively, these results implicate PKCθ-dependent destabilization of Mcl-1 as a mechanism contributing to hepatocyte lipoapoptosis.Current evidence suggests that hepatic steatosis is present in up to 30% of the American population (1). A subset of these individuals develop severe hepatic lipotoxicity, a syndrome referred to as NASH² (2), which can progress to cirrhosis and its chronic sequela (3, 4). A major risk factor for hepatic lipotoxicity is insulin resistance (5–7), resulting in excessive lipolysis within peripheral adipose tissue with release of high levels of free fatty acids (FFA) to the circulation. Circulating FFA are taken up by the liver via fatty acid transporter 5 and CD36 (8–10), and the bulk of hepatic neutral fat is derived from re-esterification of circulating FFA (8). Current concepts indicate that FFA, and not their esterified product (triglyceride), mediate hepatic lipotoxicity (11, 12). Elevated serum FFA correlate with liver disease severity (13–15), and therapies that enhance insulin sensitivity ameliorate hepatic lipotoxicity, in part, by decreasing plasma FFA (16). Hepatic FFA also accumulate in experimental steatohepatitis, further supporting a role for these nutrients in hepatic lipotoxicity (17). Saturated FFA are more strongly implicated in hepatic lipotoxicity than unsaturated FFA (18, 19). Saturated FFA induce hepatocyte apoptosis (20, 21), a cardinal feature of nonalcoholic fatty liver disease (22), and serum biomarkers of apoptosis are useful for identifying hepatic lipotoxicity (23). Thus, FFA-mediated lipotoxicity occurs, in part, by apoptosis.Apoptosis is regulated by members of the Bcl-2 protein family (24). These proteins can be categorized into three subsets as follows: the guardians or anti-apoptotic members of this family, which include Bcl-2, A1, Mcl-1, Bcl-x_L, and Bcl-w; the multidomain executioners or proapoptotic members of this family, which include Bax and Bak; and the messengers or biosensors of cell death, which share only the third Bcl-2 homology domain and are referred to as BH3-only proteins. This last group of proteins includes Bid, Bim, Bmf, Puma, Noxa, Hrk, Bad, and Bik. We have previously reported that cytotoxic FFA induce Bim expression by a FoxO3a-dependent mechanism that contributes, in part, to lipoapoptosis by activating Bax (20, 21). However, Bax activation can be held in check by anti-apoptotic members of the Bcl-2 family suggesting their function may also be dysregulated during FFA-mediated cytotoxicity.Bcl-2 is not expressed in hepatocytes at the protein level (25), whereas Bcl-w and Bfl-1/A1 knock-out mice have no liver phenotype (26–28). However, both potent anti-apoptotic proteins Bcl-x_L and Mcl-1 are expressed by hepatocytes and exhibit a liver phenotype in knock-out mice (29, 30), whereas up-regulation of Mcl-1 renders hepatocytes resistant to apoptosis (31–33). It has also been posited that cellular elimination of Mcl-1 is a critical step in certain proapoptotic cascades (34, 35). Mcl-1 is unique among Bcl-2 proteins in that it has a short half-life, 30–120 min in most cell types, due to the presence of two sequences rich in proline, glutamic acid, serine, and threonine, which target the protein for rapid degradation by the proteasome (36). Proteasomal degradation of Mcl-1 is promoted by ubiquitination, which in turn is regulated by various kinase cascades (36). Despite its potential importance, a role for Mcl-1 in regulating hepatocyte FFA-mediated lipoapoptosis remains unexplored.Given that FFA induce insulin resistance (37), the kinases potentially regulating lipoapoptosis are likely those also identified in insulin resistance syndromes, especially the novel PKC isoforms PKCδ and PKCθ (38). The novel PKC isoforms are activated by diacylglycerol, which rises in the presence of FFA (39–41), and diacylglycerol levels are significantly increased in NASH (42). A role for PKCδ in apoptosis has not been described. PKCθ has recently been shown to be activated by endoplasmic reticulum stress in liver cells (43) and lipids in vivo (44, 45). Furthermore, PKCθ has also been implicated in apoptosis of Jurkat cells, neuroblastoma cells, and myeloid leukemia cells (46, 47). However, neither its role in mediating lipoapoptosis nor modulating levels/activity of Bcl-2 proteins has been examined.This study addresses the role of Mcl-1 and PKCθ in FFA-induced lipoapoptosis. We identify a pathway that involves PKCθ-dependent proteasomal degradation of Mcl-1. Using inhibitors of various steps along this pathway, along with Mcl-1 mutants that are resistant to proteasomal degradation or Ser¹⁵⁹ phosphorylation, our studies implicate Mcl-1 degradation via a PKCθ-dependent process as a critical step in lipoapoptosis.