Rotenone Enhances the Antifungal Properties of Staurosporine

We studied staurosporine-induced cell death in the filamentous fungus Neurospora crassa. The generation of reactive oxygen species during the process appears to be an important signaling event, since addition of the antioxidant glutathione prevents the effects of staurosporine on fungal growth. Selected mutants with mutations in respiratory chain complex I are extremely sensitive to the drug, stressing the involvement of complex I in programmed cell death. Following this finding, we determined that the complex I-specific inhibitor rotenone used in combination with staurosporine results in a synergistic and specific antifungal activity, likely through a concerted action on intracellular glutathione depletion. Paradoxically, the synergistic antifungal activity of rotenone and staurosporine is observed in N. crassa complex I mutants and in Saccharomyces cerevisiae, which lacks complex I. In addition, it is not observed when other complex I inhibitors are used instead of rotenone. These results indicate that the rotenone effect is independent of complex I inhibition. The combination of rotenone and staurosporine is effective against N. crassa as well as against the common pathogens Aspergillus fumigatus and Candida albicans, pointing to its usefulness as an antifungal agent.Programmed cell death (PCD) refers to a genetically controlled process of cellular suicide initiated by endogenous or extrinsic signals. Many of the genes involved are widely conserved from unicellular to multicellular organisms (46). Apoptosis and autophagy, with its particular characteristics, have been recognized as the main categories of PCD (27). The process of PCD is crucial for the development and homeostasis of metazoan organisms and has been implicated in a number of human disorders, including cancer and neurodegenerative and infectious diseases (3, 10, 25, 55).The participation in PCD of mitochondria, the cellular organelles responsible for the production of most cellular ATP in eukaryotes (30), has been well established. Particularly, these organelles have a central role in the intrinsic (mitochondrion-dependent) pathway of apoptosis, which includes production of reactive oxygen species (ROS), membrane depolarization, ultrastructural changes, and the release of cytochrome c and other proteins (18, 50, 55). Drugs like staurosporine (STS), an inhibitor of protein kinases, have been used to induce the mitochondrion-dependent pathway of apoptosis (24, 35). Staurosporine (48) and derivatives have been used in clinical trials for cancer therapy (63). The complex I inhibitor rotenone too has been widely used to induce PCD and also extensively applied as a pesticide (11, 39, 56). Thus, these types of drugs can be employed for the acquisition of fundamental knowledge and for more practical applications, like modulation of the progression of PCD.Modulation of PCD by targeting metabolic pathways involved in the process can be exploited to the benefit of human health in several very significant situations, from cancer therapy (4, 57) to the treatment of fungal infections (3, 52). However, the molecular basis of PCD involves complex metabolic networks (32, 38, 59), and further work is required for their identification. Neurospora crassa has many advantages for biochemical and genetic experiments (14, 17, 23) and is thus a good model organism for the study of mechanisms of PCD. We are interested in identifying the cell molecular pathways associated with PCD and using this knowledge to devise strategies to modulate the process. In this work, we analyze the effects of STS on the N. crassa wild type and mitochondrial complex I mutants and describe the synergistic effect of combining STS and rotenone on this organism and other human-pathogenic fungi.     

Peroxidase Mechanism of Lipid-dependent Cross-linking of Synuclein with Cytochrome c: PROTECTION AGAINST APOPTOSIS VERSUS DELAYED OXIDATIVE STRESS IN PARKINSON DISEASE*

Damage of presynaptic mitochondria could result in release of proapoptotic factors that threaten the integrity of the entire neuron. We discovered that α-synuclein (Syn) forms a triple complex with anionic lipids (such as cardiolipin) and cytochrome c, which exerts a peroxidase activity. The latter catalyzes covalent hetero-oligomerization of Syn with cytochrome c into high molecular weight aggregates. Syn is a preferred substrate of this reaction and is oxidized more readily than cardiolipin, dopamine, and other phenolic substrates. Co-localization of Syn with cytochrome c was detected in aggregates formed upon proapoptotic stimulation of SH-SY5Y and HeLa cells and in dopaminergic substantia nigra neurons of rotenone-treated rats. Syn-cardiolipin exerted protection against cytochrome c-induced caspase-3 activation in a cell-free system, particularly in the presence of H₂O₂. Direct delivery of Syn into mouse embryonic cells conferred resistance to proapoptotic caspase-3 activation. Conversely, small interfering RNA depletion of Syn in HeLa cells made them more sensitive to dopamine-induced apoptosis. In human Parkinson disease substantia nigra neurons, two-thirds of co-localized Syn-cytochrome c complexes occurred in Lewy neurites. Taken together, these results indicate that Syn may prevent execution of apoptosis in neurons through covalent hetero-oligomerization of cytochrome c. This immediate protective function of Syn is associated with the formation of the peroxidase complex representing a source of oxidative stress and postponed damage.Lewy bodies (LBs),3 mitochondrial impairment, and oxidative stress are cardinal features of Parkinson disease (PD) and several related neurodegenerative disorders (1, 2). Aggregation of α-synuclein (Syn), an abundant protein in synaptic terminals, plays a major role in the formation of LBs (3, 4). Neither the mechanisms of LB production nor their pathogenic or protective roles in neurodegeneration are well understood.In nigrostriatal dopaminergic synaptic terminals, mitochondria, harboring a host of death-initiating factors, are in peril of damage by reactive oxygen species generated by disrupted electron transport and/or oxidative metabolism of dopamine (DA). Because cytochrome c (cyt c)-dependent formation of apoptosomes and activation of caspases designates a point of no return in the apoptotic program, release of proapoptotic factors from synaptic mitochondria could threaten the integrity of the entire neuron. How neurons protect themselves against inadvertent release of death signals from damaged synaptic mitochondria is not known.The N-terminal fragment of Syn contains six variants of an 11-amino acid consensus motif that include an apolipoprotein-like class A2 helix participating in binding of different lipids, particularly anionic phospholipids (5). This domain is believed to be important for Syn functions in regulation of neuronal lipid metabolism, particularly turnover of a mitochondria-specific phospholipid, cardiolipin (CL) (6). However, the relevance of the Syn lipid binding capacity in regulating neuronal injury (apoptotic) responses has not been established.It is believed that oxidative stress participates in the accumulation of LB and Lewy neurites (LN) through yet to be identified pathways (7). Reportedly, Syn is co-localized with cyt c in LBs (8), indicating a potential interaction between the two proteins. Because cyt c is a redox-active hemeprotein (9, 10), its presence in the LBs in conjunction with Syn may also provide a mechanistic link of LBs with oxidative stress. We have recently reported that cyt c interacts with CL in mitochondria early in apoptosis and with phosphatidylserine (PS) in the plasma membrane after its release into the cytosol (11, 12). In both cases, this results in redox activation of cyt c and the production of complexes with high peroxidase activity that effectively catalyze peroxidation of the respective phospholipids (13).Based on these facts, we hypothesize and provide experimental evidence that Syn acts as a sacrificial scavenger of cytosolic cyt c inadvertently released from synaptic mitochondria to prevent its migration into the soma, i.e. spread of the proapoptotic signal and cell death. This vital function is realized through the emergence of a peroxidase activity of the cyt c-Syn-phospholipid complex that cross-links its components and yields covalently conjugated protein-lipid hetero-oligomers. The latter maintain lingering peroxidase activity. Thus protection against acute apoptotic cell death comes with a penalty of Syn-cyt c aggregation into a peroxidase complex capable of inducing protracted oxidative stress. Our results present a novel biochemical mechanism likely involved in Lewy body formation and explain a known paradox of a dual protective and deleterious role that Syn plays in neuronal cells.     

Structure of the c14 Rotor Ring of the Proton Translocating Chloroplast ATP Synthase

The structure of the membrane integral rotor ring of the proton translocating F₁F₀ ATP synthase from spinach chloroplasts was determined to 3.8 Å resolution by x-ray crystallography. The rotor ring consists of 14 identical protomers that are symmetrically arranged around a central pore. Comparisons with the c₁₁ rotor ring of the sodium translocating ATPase from Ilyobacter tartaricus show that the conserved carboxylates involved in proton or sodium transport, respectively, are 10.6–10.8 Å apart in both c ring rotors. This finding suggests that both ATPases have the same gear distance despite their different stoichiometries. The putative proton-binding site at the conserved carboxylate Glu⁶¹ in the chloroplast ATP synthase differs from the sodium-binding site in Ilyobacter. Residues adjacent to the conserved carboxylate show increased hydrophobicity and reduced hydrogen bonding. The crystal structure reflects the protonated form of the chloroplast c ring rotor. We propose that upon deprotonation, the conformation of Glu⁶¹ is changed to another rotamer and becomes fully exposed to the periphery of the ring. Reprotonation of Glu⁶¹ by a conserved arginine in the adjacent a subunit returns the carboxylate to its initial conformation.ATP synthases found in the energy-transducing membranes of bacteria, mitochondria, and chloroplasts catalyze ATP synthesis and ATP hydrolysis coupled with transmembrane proton or sodium ion transport. The enzymes are multi-subunit complexes composed of an extra-membranous catalytic F₁ domain and an interconnected integral membrane F₀ domain. The hydrophilic F₁ domain consists of five different polypeptides with a stoichiometry of α₃β₃γδϵ. Detailed structural information obtained with the mitochondrial enzyme (1–3) in combination with biochemical (4), biophysical (5), and single molecule studies (6–9) revealed that synthesis or hydrolysis of ATP in the F₁ domain is accomplished via a rotary catalytic mechanism. In addition to information on the catalytic mechanism, structure analysis and single molecule studies of the mitochondrial or the chloroplast F₁ complex have also unraveled the molecular mechanism of several F₁-specific inhibitors (10–14). Less detailed information is available on the integral membrane F₀ domain, which consists of three different polypeptides (a, b, and c) and mediates the transfer of protons or sodium ions across the membrane. Subunits a and b were shown to reside at the periphery of a cylindrical complex formed by multiple copies of the c subunit (15–18). The number of c subunits in the cylindrical subcomplex shows substantial variation in different organisms. Ten protomers are found in ATP synthases from yeast, Escherichia coli and Bacillus PS3 (19–21), 11 in Ilyobacter tartaricus, Propionigenium modestum, and Clostridium paradoxum (22–24), 13 in the thermoalkalophilic Bacillus TA2.TA1 (25), 14 in spinach chloroplasts (26), and 15 in the cyanobacterium Spirulina platensis (27). The structure of isolated subunits a, b, and c from E. coli has been studied by mutagenesis analysis and by NMR spectroscopy in a mixed solvent that was suggested to mimic the membrane environment (28–32). These studies showed that subunit a folds with five membrane-spanning helices. The fourth of these helices directly interacts with subunit c and contains a conserved arginine (Arg²¹⁰), which is thought to be involved in proton transfer (33). Subunit b, which is present in two copies in the intact F₀, contains a single transmembrane helix. Cross-linking data support a direct interaction of the two copies of the b subunit (29). Subunit c was studied at two different pH values to obtain the protonated and deprotonated form of a conserved carboxylate (Asp⁶¹ in E. coli) that was shown to be essential for proton transport (34). NMR spectroscopy revealed that the isolated c subunit consists of two long hydrophobic membrane spanning segments connected by a short hydrophilic loop (30, 35). This loop is located close to the γ and ϵ subunit on the F₁ side of the membrane (36, 37). Low resolution x-ray crystallography, cryo-electron microscopy, and atomic force microscopy showed that the membrane-spanning helices of the multiple copies of subunit c in the intact F₀ complex are tightly packed in two concentric rings (19, 22, 26). Atomic resolution of the c ring was recently provided for the Na⁺-translocating F-type ATPase from I. tartaricus (38) and the related Na⁺-translocating V-type ATPase from Enterococcus hirae (39). Rotation of the c ring was demonstrated by cross-linking (18), fluorescence studies (40), and single molecule visualization (41, 42). Based on the structural and biochemical information on F₁ and F₀, different mechanical models have been proposed describing how the rotation of the c ring is coupled to the rotation of the F₁ rotor subunits. This rotation in turn drives sequential conformational shifts at the three catalytic β subunits that result in ATP synthesis (43–45). Vice versa hydrolysis of ATP in the F₁ domain is thought to drive rotation of the γϵc_10–15 subcomplex and transports protons or sodium ions across the membrane.Here we describe the crystal structure of the chloroplast c₁₄ rotor, which is the first structure of an isolated c ring rotor from a proton driven ATPase. The structure was solved by molecular replacement using a tetradecameric search model that was generated from a monomer taken from the I. tartaricus c₁₁ structure. The imposition of noncrystallographic symmetry restraints during refinement substantially improved electron density and structure determination.     

Aqueous Accessibility to the Transmembrane Regions of Subunit c of the Escherichia coli F1F0 ATP Synthase

Rotary catalysis in F₁F₀ ATP synthase is powered by proton translocation through the membrane-embedded F₀ sector. Proton binding and release occur in the middle of the membrane at Asp-61 on transmembrane helix (TMH) 2 of subunit c. Previously the reactivity of Cys substituted into TMH2 revealed extensive aqueous access at the cytoplasmic side as probed with Ag⁺ and other thiolate-directed reagents. The analysis of aqueous accessibility of membrane-embedded regions in subunit c was extended here to TMH1 and the periplasmic side of TMH2. The Ag⁺ sensitivity of Cys substitutions was more limited on the periplasmic versus cytoplasmic side of TMH2. In TMH1, Ag⁺ sensitivity was restricted to a pocket of four residues lying directly behind Asp-61. Aqueous accessibility was also probed using Cd²⁺, a membrane-impermeant soft metal ion with properties similar to Ag⁺. Cd²⁺ inhibition was restricted to the I28C substitution in TMH1 and residues surrounding Asp-61 in TMH2. The overall pattern of inhibition, by all of the reagents tested, indicates highest accessibility on the cytoplasmic side of TMH2 and in a pocket of residues around Asp-61, including proximal residues in TMH1. Additionally subunit a was shown to mediate access to this region by the membrane-impermeant probe 2-(trimethylammonium)ethyl methanethiosulfonate. Based upon these results and other information, a pocket of aqueous accessible residues, bordered by the peripheral surface of TMH4 of subunit a, is proposed to extend from the cytoplasmic side of cTMH2 to Asp-61 in the center of the membrane.F₁F₀ ATP synthase utilizes the energy stored in an H⁺ or Na⁺ electrochemical gradient to synthesize ATP in bacteria, mitochondria, and chloroplasts (1–4). The ATP synthase complex is composed of two sectors, i.e. a water-soluble F₁ sector that is bound to a membrane-embedded F₀ sector. In bacteria, F₁ is composed of five subunits in an α₃β₃γδϵ ratio and contains three catalytic sites for ATP synthesis and/or hydrolysis centered at the α-β subunit interfaces. F₀ is composed of three subunits in an a₁b₂c_10–15 ratio and functions as the ion-conducting pathway (5–9). Ion translocation through F₀ drives rotation of a cylindrical ring of c-subunits that is coupled to rotation of the γ subunit within the (αβ)₃ hexamer of F₁ to force conformational changes in the three active sites and in turn drive synthesis of ATP by the binding change mechanism (1–4, 10–13).Subunit c of F₀ folds in the membrane as a hairpin of two extended α-helices. In Escherichia coli, 10 copies of subunit c pack together to form a decameric ring with TMH1² on the inside and TMH2 on the periphery (6, 14). An atomic resolution structure of the Na⁺-translocating c₁₁-ring from Ilyobacter tartaricus was recently published by Meier et al. (8). In the c₁₁ structure, the Na⁺ binding site is formed by two interacting c subunits. The essential Na⁺-binding Glu residue, which corresponds to Asp-61 in E. coli, is located in TMH2 at the middle of the lipid bilayer. Subunit a consists of five transmembrane helices, four of which likely interact as a four-helix bundle (15–18). Subunit a lies on the periphery of the c-ring with TMHs 4 and 5 from subunit a and TMH2 from subunit c forming the a-c interface (18–21). During ion translocation through F₀, the essential Arg-210 on TMH4 of subunit a is postulated to facilitate the protonation/deprotonation cycle at Asp-61 of subunit c and cause the rotation of the c-ring past the stationary subunit a (3, 4, 19).Chemical modification of cysteine-substituted transmembrane proteins has been widely used as a means of probing the aqueous accessible regions (22–24). The reactivity of a substituted cysteine to thiolate-directed probes provides an indication of aqueous accessibility because the reactive thiolate species is preferentially formed in an aqueous environment. The aqueous accessibility of the five TMHs in subunit a of E. coli F₀ has been probed using Ag⁺ and NEM (19, 25–27). The results suggest the presence of an aqueous accessible channel in subunit a in the center of TMHs 2–5 extending from the periplasm to the center of the membrane. Protons entering through this periplasmic access channel are postulated to bind to the essential Asp-61 residues of the c-ring and exit to the cytoplasm by a still uncertain pathway at the peripheral face of aTMH4 with protonation/deprotonation of Asp-61 driving c-ring rotation.During H⁺-driven ATP synthesis, two models for the pathway by which H⁺ or Na⁺ exit to the cytoplasm have been proposed. The first model proposes that the ions bound at Asp-61 exit to the cytoplasm via a half-channel composed at least partially by residues in TMH4 of subunit a (25–27). Chemical modification studies of Cys-substituted subunit a of E. coli revealed an aqueous accessible surface of TMH4 that includes the essential Arg-210 residue, which extended from the center of the membrane to the cytoplasm, suggesting that the ion exit channel may lie at the a-c interface (19, 25). Alternatively studies of the c-ring from the I. tartaricus enzyme indicate that Na⁺ can access Glu-65 in the absence of other F₀ subunits, suggesting an intrinsic channel in subunit c (28, 29). However, no such channel was apparent in the crystal structure of the c₁₁-ring (8). In a previous study (30), we probed the thiolate reactivity of Cys substitutions in the cytoplasmic half of TMH2 in subunit c. These experiments revealed extensive reactivity to sulfhydryl-directed reagents on the peripheral face of cTMH2, supporting the presence of the cytoplasmic exit channel at the a-c interface. In this study, we extended the survey of aqueous accessibility in transmembrane regions by probing thiolate reactivity of Cys substitutions in TMH1 and in the periplasmic half of TMH2. The reactivity of Cys substituted into these regions proved to be more limited. Only a small region of TMH1, lying directly behind Asp-61, was reactive with Ag⁺. In addition to Ag⁺, we used Cd²⁺ as a complementary, membrane-impermeant probe for aqueous accessibility. The survey of Cd²⁺ sensitivity confirmed that aqueous accessibility from the cytoplasm is much greater for residues packing at the periphery of the c-ring. The experiments reported here distinguish the aqueous accessible and inaccessible regions of the c-ring and strengthen evidence that the cytoplasmic H⁺ exit channel is situated at the a-c interface.     

The Cytochrome c Maturation Operon Is Involved in Manganese Oxidation in Pseudomonas putida GB-1

A Pseudomonas putida strain, strain GB-1, oxidizes Mn²⁺ to Mn oxide in the early stationary growth phase. It also secretes a siderophore (identified as pyoverdine) when it is subjected to iron limitation. After transposon (Tn5) mutagenesis several classes of mutants with differences in Mn²⁺ oxidation and/or secretion of the Mn²⁺-oxidizing activity were identified. Preliminary analysis of the Tn5 insertion site in one of the nonoxidizing mutants suggested that a multicopper oxidase-related enzyme is involved in Mn²⁺ oxidation. The insertion site in another mutant was preliminarily identified as a gene involved in the general protein secretion pathway. Two mutants defective in Mn²⁺-oxidizing activity also secreted porphyrins into the medium and appeared to be derepressed for pyoverdine production. These strains were chosen for detailed analysis. Both mutants were shown to contain Tn5 insertions in the ccmF gene, which is part of the cytochrome c maturation operon. They were cytochrome oxidase negative and did not contain c-type cytochromes. Complementation with part of the ccm operon isolated from the wild type restored the phenotype of the parent strain. These results indicate that a functional ccm operon is required for Mn²⁺ oxidation in P. putida GB-1. A possible relationship between porphyrin secretion resulting from the ccm mutation and stimulation of pyoverdine production is discussed.In a number of studies during the last three decades it has been shown that various microbial species are able to stimulate the oxidation of Mn²⁺ through direct catalysis. These organisms produce proteinaceous macromolecules which catalyze the oxidation reaction. Manganese oxidations by a soil Arthrobacter species (24), Oceanospirillum and Vibrio strains (2, 3), Pseudomonas putida MnB1 (22, 30), Leptothrix discophora SS-1 (1, 11), and marine Bacillus strain SG-1 (23) are examples in which enzymes are most likely involved in the process. P. putida MnB1 produces a soluble protein which catalytically oxidizes Mn²⁺ in cell extracts (22). Manganese-oxidizing proteins from L. discophora SS-1 (1, 11) and from the spore coats of Bacillus strain SG-1 (43) have been identified on polyacrylamide gels. The oxidizing proteins have not been quantitatively purified or analyzed so far. In Bacillus strain SG-1, an operon containing seven genes appears to be involved in Mn²⁺ oxidation (46). One of these genes encodes a 137-kDa protein related to the family of multicopper oxidases (47). In a previous study we reported the isolation of a structural gene and its promoter postulated to be involved in Mn²⁺ oxidation in L. discophora (19). The encoded protein also contains the copper-binding signatures of multicopper oxidases. The oxidase-related proteins may represent Mn²⁺-oxidizing enzymes (44), but evidence supporting this hypothesis is still lacking.In this paper we describe a genetic analysis of Mn²⁺ oxidation in a freshwater Pseudomonas strain, strain GB-1. In a previous study (32) this strain was preliminarily identified as a Pseudomonas fluorescens strain, but more recent data (see Materials and Methods) indicate that it should be identified as a P. putida strain. When supplied with Mn²⁺ ions, the cells deposit manganese oxide around the outer membrane in the early stationary growth phase (32). They form brown colonies on Mn²⁺-containing agar. Experiments performed with cell extracts indicated that Mn²⁺ oxidation is catalyzed by a protein. The Mn²⁺-oxidizing factor was partially purified, and electrophoresis on an acrylamide gradient gel revealed oxidizing proteins with apparent molecular weights of ca. 250,000 and 180,000 (32). An additional oxidizing factor with a lower molecular weight (ca. 130,000) was identified in another study by using different isolation and electrophoretic procedures (16). It has been suggested that the Mn²⁺-oxidizing protein isolated is part of a larger complex which disintegrates into smaller fragments that retain activity (32). The protein is supposed to be located in the outer membrane of the bacteria. It has not been chemically characterized, and nothing is known about its cellular function or about the possible involvement of other cellular components, such as electron carriers, in Mn²⁺ oxidation.We used transposon mutagenesis to identify genes relevant to the Mn²⁺-oxidizing process in P. putida GB-1. One of these genes appeared to be part of the cytochrome c maturation operon. Transposon insertion in this gene not only abolished Mn²⁺ oxidation but also led to secretion of siderophores and porphyrins.An accompanying report on the involvement of the cytochrome c maturation operon in Mn²⁺ oxidation in P. putida MnB1 (14) supports our findings.     

A Novel Mitochondrial Sphingomyelinase in Zebrafish Cells

Sphingolipids are important signaling molecules in many biological processes, but little is known regarding their physiological roles in the mitochondrion. We focused on the biochemical characters of a novel sphingomyelinase (SMase) and its function in mitochondrial cer a mide generation in zebrafish embryonic cells. The cloned SMase cDNA encoded a polypeptide of 545 amino acid residues (putative molecular weight, 61,300) containing a mitochondrial localization signal (MLS) and a predicted transmembrane domain. The mature endogenous enzyme was predicted to have a molecular weight of 57,000, and matrix-assisted laser de sorp tion ionization time-of-flight mass spectrometry analysis indicated that the N-terminal amino acid residue of the mature enzyme was Ala-36. The purified enzyme optimally hydrolyzed [¹⁴C]sphingomyelin in the presence of 10 mm Mg²⁺ at pH 7.5. In HEK293 cells that overexpressed SMase cDNA, the enzyme was localized to the mitochondrial fraction, whereas mutant proteins lacking MLS or both the MLS and the transmembrane domain were absent from the mitochondrial fraction. Endogenous SMase protein co-localized with a mitochondrial cytostaining marker. Using a protease protection assay, we found that SMase was distributed throughout the intermembrane space and/or the inner membrane of the mitochondrion. Furthermore, the overexpression of SMase in HEK293 cells induced cer a mide generation and sphingomyelin hydrolysis in the mitochondrial fraction. Antisense phosphorothioate oligonucleotide-induced knockdown repressed cer a mide generation and sphingomyelin hydrolysis in the mitochondrial fraction in zebrafish embryonic cells. These observations indicate that SMase catalyzes the hydrolysis of sphingomyelin and generates cer a mide in mitochondria in fish cells.Sphingomyelinase (SMase,² sphingomyelin phosphodiesterase, EC 3.1.4.12) hydrolyzes sphingomyelin and produces ceramide and phosphocholine. Ceramide plays an important role as a signaling molecule in cell proliferation, apoptosis, cell cycle arrest, differentiation, and the stress response in animal cells (1–5). To date, three distinct classes of acid, neutral, and alkaline SMases have been identified according to optimum pH, cation dependence, amino acid sequence, and subcellular localization (3).The Mg²⁺-dependent neutral SMases have emerged as major candidates in the mediation of ceramide-induced cell signaling (6). Recent research has identified at least three distinct neutral SMases in human and mouse, designated as neutral SMase 1, SMase 2, and SMase 3 (7–9). Neutral SMase 1 was the first SMase identified in human and mouse. Although mammalian enzymes exhibited Mg²⁺-dependent neutral SMase activity in vitro (9), no significant biological functions in sphingomyelin and ceramide metabolism were identified in SMase 1-overexpressing cells (10) or neutral SMase 1 knock-out mice (11). In zebrafish embryos, Mg²⁺-dependent neutral SMase 1 produced ceramide and caused thalidomide-induced vascular defects (12). In addition, SMase 1 was found to mediate heat-induced ceramide generation and apoptosis (13).The neutral SMase 2 gene SMPD3, has also been identified based on its similarity to Bacillus cereus SMase DNA sequences (7). This gene encodes a membrane-bound protein expressed in the brain and liver that has two highly hydrophobic segments near the N-terminal region, both of which are thought to function as transmembrane domains. Unlike neutral SMase 1, neutral SMase 2 possesses Mg²⁺-dependent neutral SMase activity in vivo in MCF-7 cells (14). When overexpressed in the confluent phase of MCF-7 cells, mouse neutral SMase 2 was palmitoylated via thioester bonds and localized in the inner leaflet of the plasma membrane (15). In MCF-7 cells stably expressing neutral SMase 2, the enzyme inhibited cell growth and was required for cells to undergo confluence-induced cell cycle arrest (16). Interestingly, neutral SMase 2 was isolated as the confluent 3Y1 cell-associated 1 gene (cca1) in rat 3Y1 cells (17). Neutral SMase 2 has been implicated in signal transduction events in cell growth and the cellular response to cytokines (18, 19), oxidative stress (20), and amyloid β-peptide (21).Stoffel et al. (22) demonstrated that gene-targeted mice deficient for neutral SMase 2 developed a novel form of dwarfism and had delayed puberty as part of a hypothalamus-induced pituitary hormone deficiency. Strikingly, positional cloning of the recessive mutation fragilitas ossium in mice identified a deletion in the gene that encodes neutral SMase 2, leading to the complete loss of neutral SMase activity (23). The mutant fragilitas ossium mice develop severe osteogenesis and dentinogenesis imperfecta, with no collagen defect. Thus, mouse neutral SMase 2 is essential for late embryonic and postnatal development.Mitochondria contain small amounts of a variety of sphingolipids, including ceramide and sphingomyelin (24–26), which may be derived from the endoplasmic reticulum via intimate membrane contacts or produced in response to apoptosis. For mitochondria isolated from HL-60 cells, treatment with ceramide inhibited the mitochondrial respiratory chain complex III (27). Birbes et al. (28) found that the selective hydrolysis of a mitochondrial pool of sphingomyelin induced apoptosis. They transfected MCF-7 cells with B. cereus SMase targeted to various subcellular organelles, but they observed cytochrome c release and apoptosis induction only when the enzyme was targeted to the mitochondria. Ceramide activated the mitochondrial protein phosphatase 2A, which dephosphorylated Bcl-2 and led to apoptosis (29). In MCF-7 cells, mitochondrial ceramide generation in response to tumor necrosis factor-α induced Bax translocation to mitochondria and subsequent cytochrome c release and apoptosis (30). The permeability of the mitochondrial outer membrane correlates directly with the level of ceramide in the membrane (31). The concentration of ceramide at which significant channel formation occurs is consistent with the level of mitochondrial ceramide that occurs during the induction phase of apoptosis (31). In isolated mitochondria, ceramide can also form membrane channels large enough to release cytochrome c and other small proteins (32). Ceramide-metabolizing enzymes, such as a bovine liver ceramide synthase (33) and human ceramidase (34), are localized to the mitochondrion. These observations suggest the existence of a mitochondrial pool of sphingomyelin and the function of a sphingomyelin-specific metabolic pathway in mitochondria. However, no SMase has been identified in mitochondria.We identified and examined the biochemical properties of a novel SMase localized to the zebrafish mitochondrion. The enzyme was cloned from a cDNA library of embryonic zebrafish cells. It was found to regulate mitochondrial ceramide levels.     

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).     

Analysis of Free Energy Signals Arising from Nucleotide Hybridization Between rRNA and mRNA Sequences during Translation in Eubacteria

A decoding algorithm is tested that mechanistically models the progressive alignments that arise as the mRNA moves past the rRNA tail during translation elongation. Each of these alignments provides an opportunity for hybridization between the single-stranded, -terminal nucleotides of the 16S rRNA and the spatially accessible window of mRNA sequence, from which a free energy value can be calculated. Using this algorithm we show that a periodic, energetic pattern of frequency 1/3 is revealed. This periodic signal exists in the majority of coding regions of eubacterial genes, but not in the non-coding regions encoding the 16S and 23S rRNAs. Signal analysis reveals that the population of coding regions of each bacterial species has a mean phase that is correlated in a statistically significant way with species () content. These results suggest that the periodic signal could function as a synchronization signal for the maintenance of reading frame and that codon usage provides a mechanism for manipulation of signal phase.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32]     

Splitting the BLOSUM Score into Numbers of Biological Significance

Mathematical tools developed in the context of Shannon information theory were used to analyze the meaning of the BLOSUM score, which was split into three components termed as the BLOSUM spectrum (or BLOSpectrum). These relate respectively to the sequence convergence (the stochastic similarity of the two protein sequences), to the background frequency divergence (typicality of the amino acid probability distribution in each sequence), and to the target frequency divergence (compliance of the amino acid variations between the two sequences to the protein model implicit in the BLOCKS database). This treatment sharpens the protein sequence comparison, providing a rationale for the biological significance of the obtained score, and helps to identify weakly related sequences. Moreover, the BLOSpectrum can guide the choice of the most appropriate scoring matrix, tailoring it to the evolutionary divergence associated with the two sequences, or indicate if a compositionally adjusted matrix could perform better.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29]     

Algorithms for Finding Small Attractors in Boolean Networks

A Boolean network is a model used to study the interactions between different genes in genetic regulatory networks. In this paper, we present several algorithms using gene ordering and feedback vertex sets to identify singleton attractors and small attractors in Boolean networks. We analyze the average case time complexities of some of the proposed algorithms. For instance, it is shown that the outdegree-based ordering algorithm for finding singleton attractors works in time for , which is much faster than the naive time algorithm, where is the number of genes and is the maximum indegree. We performed extensive computational experiments on these algorithms, which resulted in good agreement with theoretical results. In contrast, we give a simple and complete proof for showing that finding an attractor with the shortest period is NP-hard.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32]     

Context-dependent Bcl-2/Bak Interactions Regulate Lymphoid Cell Apoptosis

The release of cytochrome c from mitochondria, which leads to activation of the intrinsic apoptotic pathway, is regulated by interactions of Bax and Bak with antiapoptotic Bcl-2 family members. The factors that regulate these interactions are, at the present time, incompletely understood. Recent studies showing preferences in binding between synthetic Bcl-2 homology domain 3 and antiapoptotic Bcl-2 family members in vitro have suggested that the antiapoptotic proteins Mcl-1 and Bcl-x_L, but not Bcl-2, restrain proapoptotic Bak from inducing mitochondrial membrane permeabilization and apoptosis. Here we show that Bak protein has a much higher affinity than the 26-amino acid Bak Bcl-2 homology domain 3 for Bcl-2, that some naturally occurring Bcl-2 allelic variants have an affinity for full-length Bak that is only 3-fold lower than that of Mcl-1, and that endogenous levels of these Bcl-2 variants (which are as much as 40-fold more abundant than Mcl-1) restrain part of the Bak in intact lymphoid cells. In addition, we demonstrate that Bcl-2 variants can, depending on their affinity for Bak, substitute for Mcl-1 in protecting cells. Thus, the ability of Bcl-2 to protect cells from activated Bak depends on two important contextual variables, the identity of the Bcl-2 present and the amount expressed.The release of cytochrome c from mitochondria, which leads to activation of the intrinsic apoptotic pathway, is regulated by Bcl-2 family members (1–5). This group of proteins consists of three subgroups: Bax and Bak, which oligomerize upon death stimulation to form a putative pore in the outer mitochondrial membrane, thereby allowing efflux of cytochrome c and other mitochondrial intermembrane space components; Bcl-2, Bcl-x_L, Mcl-1, and other antiapoptotic homologs, which antagonize the effects of Bax and Bak; and BH3-only proteins² such as Bim, Bid, and Puma, which are proapoptotic Bcl-2 family members that share only limited homology with the other two groups in a single 15-amino acid domain (the BH3 domain, see Ref. 6). Although it is clear that BH3-only proteins serve as molecular sensors of various stresses and, when activated, trigger apoptosis (3, 6–11), the mechanism by which they do so remains incompletely understood. One current model suggests that BH3-only proteins trigger apoptosis solely by binding and neutralizing antiapoptotic Bcl-2 family members, thereby causing them to release the activated Bax and Bak that are bound (reviewed in Refs. 9 and 10; see also Refs. 12 and 13), whereas another current model suggests that certain BH3-only proteins also directly bind to and activate Bax (reviewed in Ref. 3; see also Refs. 14–17). Whichever model turns out to be correct, both models agree that certain antiapoptotic Bcl-2 family members can inhibit apoptosis, at least in part, by binding and neutralizing activated Bax and Bak before they permeabilize the outer mitochondrial membrane (13, 18, 19).Much of the information about the interactions between pro- and antiapoptotic Bcl-2 family members has been derived from the study of synthetic peptides corresponding to BH3 domains. In particular, these synthetic peptides have been utilized as surrogates for the full-length proapoptotic proteins during structure determinations (20–22) as well as in functional studies exploring the effect of purified BH3 domains on isolated mitochondria (14, 23) and on Bax-mediated permeabilization of lipid vesicles (15).Recent studies using these same peptides have suggested that interactions of the BH3 domains of Bax, Bak, and the BH3-only proteins with the “BH3 receptors” of the antiapoptotic Bcl-2 family members are not all equivalent. Surface plasmon resonance, a technique that is widely used to examine the interactions of biomolecules under cell-free conditions (24–26), has demonstrated that synthetic BH3 peptides of some BH3-only family members show striking preferences, with the Bad BH3 peptide binding to Bcl-2 and Bcl-x_L but not Mcl-1, and the Noxa BH3 peptide binding to Mcl-1 but not Bcl-2 or Bcl-x_L (27). Likewise, the Bak BH3 peptide exhibits selectivity, with high affinity for Bcl-x_L and Mcl-1 but not Bcl-2 (12). The latter results have led to a model in which Bcl-x_L and Mcl-1 restrain Bak and inhibit Bak-dependent apoptosis, whereas Bcl-2 does not (10).Because the Bak protein contains multiple recognizable domains in addition to its BH3 motif (28, 29), we compared the binding of Bak BH3 peptide and Bak protein to Bcl-2. Surface plasmon resonance demonstrated that Bcl-2 binds Bak protein with much higher affinity than the Bak 26-mer BH3 peptide. Further experiments demonstrated that the K_D for Bak differs among naturally occurring Bcl-2 sequence variants but is only 3-fold higher than that of Mcl-1 in some cases. In light of previous reports that Bcl-2 overexpression contributes to neoplastic transformation (30–33) and drug resistance (34–36) in lymphoid cells, we also examined Bcl-2 expression and Bak binding in a panel of neoplastic lymphoid cell lines. Results of these experiments demonstrated that Bcl-2 expression varies among different lymphoid cell lines but is up to 40-fold more abundant than Mcl-1. In lymphoid cell lines with abundant Bcl-2, Bak is detected in Bcl-2 as well as Mcl-1 immunoprecipitates; and Bak-dependent apoptosis induced by Mcl-1 down-regulation can be prevented by Bcl-2 overexpression. Collectively, these observations shed new light on the role of Bcl-2 in binding and neutralizing Bak.     

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.