Expression of divergent methyl/alkyl coenzyme M reductases from uncultured archaea.

Methanogens and anaerobic methane-oxidizing archaea (ANME) are important players in the global carbon cycle. Methyl-coenzyme M reductase (MCR) is a key enzyme in methane metabolism, catalyzing the last step in methanogenesis and the first step in anaerobic methane oxidation. Divergent mcr and mcr-like genes have recently been identified in uncultured archaeal lineages. However, the assembly and biochemistry of MCRs from uncultured archaea remain largely unknown. Here we present an approach to study MCRs from uncultured archaea by heterologous expression in a methanogen, Methanococcus maripaludis. Promoter, operon structure, and temperature were important determinants for MCR production. Both recombinant methanococcal and ANME-2 MCR assembled with the host MCR forming hybrid complexes, whereas tested ANME-1 MCR and ethyl-coenzyme M reductase only formed homogenous complexes. Together with structural modeling, this suggests that ANME-2 and methanogen MCRs are structurally similar and their reaction directions are likely regulated by thermodynamics rather than intrinsic structural differences.

Dissolved organic phosphorus utilization by the marine bacterium Ruegeria pomeroyi DSS-3 reveals chain length-dependent polyphosphate degradation.

Dissolved organic phosphorus (DOP) is a critical nutritional resource for marine microbial communities. However, the relative bioavailability of different types of DOP, such as phosphomonoesters (P-O-C) and phosphoanhydrides (P-O-P), is poorly understood. Here we assess the utilization of these P sources by a representative bacterial copiotroph, Ruegeria pomeroyi DSS-3. All DOP sources supported equivalent growth by R. pomeroyi, and all DOP hydrolysis rates were upregulated under phosphorus depletion (-P). A long-chain polyphosphate (45polyP) showed the lowest hydrolysis rate of all DOP substrates tested, including tripolyphosphate (3polyP). Yet the upregulation of 45polyP hydrolysis under -P was greater than any other substrate analyzed. Proteomics revealed three common P acquisition enzymes potentially involved in polyphosphate utilization, including two alkaline phosphatases, PhoD and PhoX, and one 5'-nucleotidase (5'-NT). Results from DOP substrate competition experiments show that these enzymes likely have broad substrate specificities, including chain length-dependent reactivity toward polyphosphate. These results confirm that DOP, including polyP, are bioavailable nutritional P sources for R. pomeroyi, and possibly other marine heterotrophic bacteria. Furthermore, the chain-length dependent mechanisms, rates and regulation of polyP hydrolysis suggest that these processes may influence the composition of DOP and the overall recycling of nutrients within marine dissolved organic matter.

Identification of lipidomic profiles associated with drug-resistant prostate cancer cells.

BACKGROUND: The association of circulating lipids with clinical outcomes of drug-resistant castration-resistant prostate cancer (DR-CRPC) is not fully understood. While it is known that increases in select lipids correlate to decreased survival, neither the mechanisms mediating these alterations nor the correlation of resistance to drug treatments is well characterized. METHODS: This gap-in-knowledge was addressed using in vitro models of non-cancerous, hormone-sensitive, CRPC and drug-resistant cell lines combined with quantitative LC-ESI-Orbitrap-MS (LC-ESI-MS/MS) lipidomic analysis and subsequent analysis such as Metaboanalyst and Lipid Pathway Enrichment Analysis (LIPEA). RESULTS: Several lipid regulatory pathways were identified that are associated with Docetaxel resistance in prostate cancer (PCa). These included those controlling glycerophospholipid metabolism, sphingolipid signaling and ferroptosis. In total, 7460 features were identified as being dysregulated between the cell lines studied, and 21 lipid species were significantly altered in drug-resistant cell lines as compared to nonresistant cell lines. Docetaxel resistance cells (PC3-Rx and DU145-DR) had higher levels of phosphatidylcholine (PC), oxidized lipid species, phosphatidylethanolamine (PE), and sphingomyelin (SM) as compared to parent control cells (PC-3 and DU-145). Alterations were also identified in the levels of phosphatidic acid (PA) and diacylglyceride (DAG), whose levels are regulated by Lipin (LPIN), a phosphatidic acid phosphatase that converts PA to DAG. Data derived from cBioPortal demonstrated a population of PCa patients expressing mutations aligning with amplification of LPIN1, LPIN2 and LPIN3 genes. Lipin amplification in these genes correlated to decreased survival in these patients. Lipin-1 mRNA expression also showed a similar trend in PCa patient data. Lipin-1, but not Lipin-2 or - 3, was detected in several prostate cancer cells, and was increased in 22RV1 and PC-3 cell lines. The increased expression of Lipin-1 in these cells correlated with the level of PA. CONCLUSION: These data identify lipids whose levels may correlate to Docetaxel sensitivity and progression of PCa. The data also suggest a correlation between the expression of Lipin-1 in cells and patients with regards to prostate cancer cell aggressiveness and patient survivability. Ultimately, these data may be useful for identifying markers of lethal and/or metastatic prostate cancer.

Burkholderia thailandensis outer membrane vesicles exert antimicrobial activity against drug-resistant and competitor microbial species.

Gram-negative bacteria secrete outer membrane vesicles (OMVs) that play critical roles in intraspecies, interspecies, and bacteria-environment interactions. Some OMVs, such as those produced by Pseudomonas aeruginosa, have previously been shown to possess antimicrobial activity against competitor species. In the current study, we demonstrate that OMVs from Burkholderia thailandensis inhibit the growth of drug-sensitive and drug-resistant bacteria and fungi. We show that a number of antimicrobial compounds, including peptidoglycan hydrolases, 4-hydroxy-3-methyl-2-(2-non-enyl)-quinoline (HMNQ) and long-chain rhamnolipid are present in or tightly associate with B. thailandensis OMVs. Furthermore, we demonstrate that HMNQ and rhamnolipid possess antimicrobial and antibiofilm properties against methicillin-resistant Staphylococcus aureus (MRSA). These findings indicate that B. thailandensis secretes antimicrobial OMVs that may impart a survival advantage by eliminating competition. In addition, bacterial OMVs may represent an untapped resource of novel therapeutics effective against bio-film-forming and multidrug-resistant organisms.

Posttranslational Methylation of Arginine in Methyl Coenzyme M Reductase Has a Profound Impact on both Methanogenesis and Growth of Methanococcus maripaludis.

Catalyzing the key step for anaerobic production and/or oxidation of methane and likely other short-chain alkanes, methyl coenzyme M reductase (Mcr) and its homologs play a key role in the global carbon cycle. The McrA subunit possesses up to five conserved posttranslational modifications (PTMs) at its active site. It was previously suggested that methanogenesis marker protein 10 (Mmp10) could play an important role in methanogenesis. To systematically examine its physiological role, mmpX (locus tag MMP1554), the gene encoding Mmp10 in Methanococcus maripaludis, was deleted with a new genetic tool, resulting in the complete loss of the 5-C-(S)-methylarginine PTM of residue 275 in the McrA subunit. When the ΔmmpX mutant was complemented with the wild-type gene expressed by either a strong or a weak promoter, methylation was fully restored. Compared to the parental strain, maximal rates of methane formation by whole cells were reduced by 40 to 60% in the ΔmmpX mutant. The reduction in activity was fully reversed by the complement with the strong promoter. Site-directed mutagenesis of mmpX resulted in a differential loss of arginine methylation among the mutants in vivo, suggesting that activities of Mmp10 directly modulated methylation. R275 was present in a highly conserved PXRR275(A/S)R(G/A) signature sequence in McrAs. The only other protein in M. maripaludis containing a similar sequence was not methylated, suggesting that Mmp10 is specific for McrA. In conclusion, Mmp10 modulates the methyl-Arg PTM on McrA in a highly specific manner, which has a profound impact on Mcr activity.IMPORTANCE Mcr is the key enzyme in methanogenesis and a promising candidate for bioengineering the conversion of methane to liquid fuel. Our knowledge of Mcr is still limited. In terms of complexity, uniqueness, and environmental importance, Mcr is more comparable to photosynthetic reaction centers than conventional enzymes. PTMs have long been hypothesized to play key roles in modulating Mcr activity. Here, we directly link the mmpX gene to the arginine PTM of Mcr, demonstrate its association with methanogenesis activity, and offer insights into its substrate specificity and putative cofactor binding sites. This is also the first time that a PTM of McrA has been shown to have a substantial impact on both methanogenesis and growth in the absence of additional stressors.

Assembly of Methyl Coenzyme M Reductase in the Methanogenic Archaeon Methanococcus maripaludis.

Methyl coenzyme M reductase (MCR) is a complex enzyme that catalyzes the final step in biological methanogenesis. To better understand its assembly, the recombinant MCR from the thermophile Methanothermococcus okinawensis (rMCRok) was expressed in the mesophile Methanococcus maripaludis The rMCRok was posttranslationally modified correctly and contained McrD and the unique nickel tetrapyrrole coenzyme F430 Subunits of the native M. maripaludis (MCRmar) were largely absent, suggesting that the recombinant enzyme was formed by an assembly of cotranscribed subunits. Strong support for this hypothesis was obtained by expressing a chimeric operon comprising the His-tagged mcrA from M. maripaludis and the mcrBDCG from M. okinawensis in M. maripaludis The His-tagged purified rMCR then contained the M. maripaludis McrA and the M. okinawensis McrBDG. The present study prompted us to form a working model for MCR assembly, which can be further tested by the heterologous expression system established here.IMPORTANCE Approximately 1.6% of the net primary production of plants, algae, and cyanobacteria are processed by biological methane production in anoxic environments. This accounts for about 74% of the total global methane production, up to 25% of which is consumed by anaerobic oxidation of methane (AOM). Methyl coenzyme M reductase (MCR) is the key enzyme in both methanogenesis and AOM. MCR is assembled as a dimer of two heterotrimers, where posttranslational modifications and F430 cofactors are embedded in the active sites. However, this complex assembly process remains unknown. Here, we established a heterologous expression system for MCR to learn how MCR is assembled.

Profiling Cellular Substrates of Lysine Acetyltransferases GCN5 and p300 with Orthogonal Labeling and Click Chemistry.

p300 and GCN5 are two representative lysine acetyltransferases (KATs) in mammalian cells. It was recently reported that they possess multiple acyltransferase activities including acetylation, propionylation, and butyrylation of the ε-amino group of lysine residues of histones and non-histone protein substrates. Although thousands of acetylated substrates and acetylation sites have been identified by mass spectrometry-based proteomic screening, our knowledge about the causative connections between individual KAT members and their corresponding sub-acylomes remain very limited. Herein, we applied 3-azidopropionyl CoA (3AZ-CoA) as a bioorthogonal surrogate of acetyl-, propionyl- and butyryl-CoA for KAT substrate identification. We successfully attached the azide as a chemical warhead to cellular substrates of wild-type p300 and engineered GCN5. The substrates were subsequently labeled with biotin tag through the copper-catalyzed azide-alkyne cycloaddition (CuAAC). Following protein enrichment on streptavidin-coated resin, we conducted LC-MS/MS studies from which more than four hundred proteins were identified as GCN5 or p300 substrate candidates. These proteins are either p300- or GCN5-unique or shared by the two KATs and are extensively involved in various biological events including gene expression, cell cycle, and cellular metabolism. We also experimentally validated two novel substrates of GCN5, that is, IQGAP1 and SMC1. These results demonstrate extensive engagement of GCN5 and p300 in cellular pathways and provide new insights into understanding their functions in specific biological processes.

Multipart Chaperone-Effector Recognition in the Type III Secretion System of Chlamydia trachomatis.

Secretion of effector proteins into the eukaryotic host cell is required for Chlamydia trachomatis virulence. In the infection process, Scc1 and Scc4, two chaperones of the type III secretion (T3S) system, facilitate secretion of the important effector and plug protein, CopN, but little is known about the details of this event. Here we use biochemistry, mass spectrometry, nuclear magnetic resonance spectroscopy, and genetic analyses to characterize this trimolecular event. We find that Scc4 complexes with Scc1 and CopN in situ at the late developmental cycle of C. trachomatis. We show that Scc4 and Scc1 undergo dynamic interactions as part of the unique bacterial developmental cycle. Using alanine substitutions, we identify several amino acid residues in Scc4 that are critical for the Scc4-Scc1 interaction, which is required for forming the Scc4·Scc1·CopN ternary complex. These results, combined with our previous findings that Scc4 plays a role in transcription (Rao, X., Deighan, P., Hua, Z., Hu, X., Wang, J., Luo, M., Wang, J., Liang, Y., Zhong, G., Hochschild, A., and Shen, L. (2009) Genes Dev. 23, 1818-1829), reveal that the T3S process is linked to bacterial transcriptional events, all of which are mediated by Scc4 and its interacting proteins. A model describing how the T3S process may affect gene expression is proposed.

The roles of phosphorylation and SHAGGY-like protein kinases in geminivirus C4 protein induced hyperplasia.

Even though plant cells are highly plastic, plants only develop hyperplasia under very specific abiotic and biotic stresses, such as when exposed to pathogens like Beet curly top virus (BCTV). The C4 protein of BCTV is sufficient to induce hyperplasia and alter Arabidopsis development. It was previously shown that C4 interacts with two Arabidopsis Shaggy-like protein kinases, AtSK21 and 23, which are negative regulators of brassinosteroid (BR) hormone signaling. Here we show that the C4 protein interacts with five additional AtSK family members. Bikinin, a competitive inhibitor of the seven AtSK family members that interact with C4, induced hyperplasia similar to that induced by the C4 protein. The Ser49 residue of C4 was found to be critical for C4 function, since: 1) mutagenesis of Ser49 to Ala abolished the C4-induced phenotype, abolished C4/AtSK interactions, and resulted in a mutant protein that failed to induce changes in the BR signaling pathway; 2) Ser49 is phosphorylated in planta; and 3) plant-encoded AtSKs must be catalytically active to interact with C4. A C4 N-myristoylation site mutant that does not localize to the plasma membrane and does not induce a phenotype, retained the ability to bind AtSKs. Taken together, these results suggest that plasma membrane associated C4 interacts with and co-opts multiple AtSKs to promote its own phosphorylation and activation to subsequently compromise cell cycle control.

Membrane vesicle production by Chlamydia trachomatis as an adaptive response.

Bacteria have evolved specific adaptive responses to cope with changing environments. These adaptations include stress response phenotypes with dynamic modifications of the bacterial cell envelope and generation of membrane vesicles (MVs). The obligate intracellular bacterium, Chlamydia trachomatis, typically has a biphasic lifestyle, but can enter into an altered growth state typified by morphologically aberrant chlamydial forms, termed persistent growth forms, when induced by stress in vitro. How C. trachomatis can adapt to a persistent growth state in host epithelial cells in vivo is not well understood, but is an important question, since it extends the host-bacterial relationship in vitro and has thus been indicated as a survival mechanism in chronic chlamydial infections. Here, we review recent findings on the mechanistic aspects of bacterial adaptation to stress with a focus on how C. trachomatis remodels its envelope, produces MVs, and the potential important consequences of MV production with respect to host-pathogen interactions. Emerging data suggest that the generation of MVs may be an important mechanism for C. trachomatis intracellular survival of stress, and thus may aid in the establishment of a chronic infection in human genital epithelial cells.

Histone H3 lysine 79 methyltransferase Dot1 is required for immortalization by MLL oncogenes.

Chimeric oncoproteins resulting from fusion of MLL to a wide variety of partnering proteins cause biologically distinctive and clinically aggressive acute leukemias. However, the mechanism of MLL-mediated leukemic transformation is not fully understood. Dot1, the only known histone H3 lysine 79 (H3K79) methyltransferase, has been shown to interact with multiple MLL fusion partners including AF9, ENL, AF10, and AF17. In this study, we utilize a conditional Dot1l deletion model to investigate the role of Dot1 in hematopoietic progenitor cell immortalization by MLL fusion proteins. Western blot and mass spectrometry show that Dot1-deficient cells are depleted of the global H3K79 methylation mark. We find that loss of Dot1 activity attenuates cell viability and colony formation potential of cells immortalized by MLL oncoproteins but not by the leukemic oncoprotein E2a-Pbx1. Although this effect is most pronounced for MLL-AF9, we find that Dot1 contributes to the viability of cells immortalized by other MLL oncoproteins that are not known to directly recruit Dot1. Cells immortalized by MLL fusions also show increased apoptosis, suggesting the involvement of Dot1 in survival pathways. In summary, our data point to a pivotal requirement for Dot1 in MLL fusion protein-mediated leukemogenesis and implicate Dot1 as a potential therapeutic target.

Development of a serum profile for healthy aging.

Increasing numbers of Americans are reaching 85 years of age or older, yet there are no reliable biomarkers to predict who will live this long. The goal of this pilot study therefore was: (1) to identify a potential serum pattern that could identify proteins involved in longevity and (2) to determine if this pattern was a marker of longevity in an independent sample of individuals. Serum samples were analyzed in three cohorts of individuals (n = 12 in each) aged 20-34, 60-74, and ≥ 90 years who participated in The Louisiana Healthy Aging Study. The 12 most abundant proteins were removed and the remaining proteins separated by two-dimensional gel electrophoresis. Gels were matched and the intensity of each spot quantified. Multivariate discriminant analysis was used to identify a serum pattern that could separate these three age cohorts. Seven protein spots were found that correctly distinguished the subjects into the three groups. However, these spots were not as successful in discriminating the ages in a second set of 15 individuals as only eight of these subjects were placed into their correct group. These preliminary results show that the proteomics approach can be used to identify potential proteins or markers that may be involved in the aging process and/or be important determinants of longevity.

Dependence of the ejection velocities of laser-ablated ions on the laser wavelength and fluence.

Drift measurements of initial ejection velocities of matrix-assisted laser desorption/ionization matrix compounds have been made as a function of ablating laser wavelength and laser fluence. For pulsed laser irradiation just above the matrix ion appearance threshold, initial ejection velocities of protonated molecular ions of an anthranilic acid target increase from ~ 1350 m/s to ~ 1640 m/s (kinetic energies of 1.3 eV and 1.9 eV, respectively) when the ablation laser wavelength is changed from 355 nm to 266 nm. Increasing the laser fluence per pulse by up to a factor of 10 above threshold results in the appearance of a slower component of the ejected ion flux. The results are interpreted by a photomechanical ejection model in which a photoexcited surface molecule instantaneously becomes larger and recoils away from the surface.

Preparation of homogenous oligosaccharide chains from glycosphingolipids.

After the discovery of glycosphingolipid (GSL) glycan detaching enzymes, Rhodococcal endoglycoceramidase (EGCase) and leech ceramide glycanase (CGase), the method for enzymatically releasing glycans from GSLs has become the method of choice for preparing intact ceramide-free oligosaccharide chains from GSLs. This paper describes (1) the preparation of the intact oligosaccharides from GM1 (II(3)NeuAcGgOse(4)Cer) and GbOse(4)Cer as examples to show the use of CGase to prepare intact glycan chains from GSLs, and (2) the specificity and detergent requirements of Rhodococcal EGCases for the release of glycan chains from different GSLs.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of oligonucleotides.

MALDI-MS is one of the most useful techniques available for determining biomolecule mass. It offers high mass accuracy, good sensitivity, simplicity, and speed. Because singly charged ions of oligonucleotides are typically observed, MALDI-MS spectra are easy to interpret. This unit presents protocols for sample preparation and purification, matrix preparation, and matrix/analyte sample preparation. It provides an introduction to the instrumentation and its calibration, and a discussion of some of the useful applications of MALDI-MS analysis in the study of oligonucleotides. This technique is typically used for 120-mer or smaller oligonucleotides.

Protein expression patterns in zebrafish skeletal muscle: initial characterization and the effects of hypoxic exposure.

Patterns of protein expression were examined in white skeletal muscle from adult zebrafish (Danio rerio). High resolution two-dimensional gel electrophoresis resolved between 300 and 400 spots with molecular masses between 20 and 120 kDa and isoelectric points between about 5 and 8. Forty spots, representing a range of protein size, charge, and abundance were excised, digested with trypsin, and subjected to matrix-assisted laser-desorption/ionisation-time of flight mass spectrometry for protein identification. Twenty-nine spots were identified, including enzymes of energy metabolism, contractile proteins, an iron transport protein, and a heat shock protein. In addition, several spots matched theoretical proteins predicted from genome sequencing. These theoretical proteins were tentatively identified by similarity to known proteins. Patterns of muscle protein expression were then measured after zebrafish were exposed to low oxygen (16 torr) for 48 h, an exposure previously shown to increase the survival of zebrafish at more severe reductions in oxygen. Exposure to low oxygen (hypoxia) did not change the general pattern of protein expression but did affect the amounts of six low abundance proteins. The relatively subtle effects of hypoxia on patterns of muscle protein expression contrasts the widespread changes previously documented in mRNA levels in this and other species of fish during hypoxic stress. The difference between protein and mRNA expression illustrates the need to integrate both measures for a more complete understanding of gene expression in fish during hypoxic exposure.

Presence of an unusual GM2 derivative, taurine-conjugated GM2, in Tay-Sachs brain.

Tay-Sachs disease (TSD) is a classical glycosphingolipid (GSL) storage disease. Although the genetic and biochemical bases for a massive cerebral accumulation of ganglioside GM2 in TSD have been well established, the mechanism for the neural dysfunction in TSD remains elusive. Upon analysis of GSLs from a variant B TS brain, we have detected a novel GSL that has not been previously revealed. We have isolated this GSL in pure form. Using NMR spectroscopy, mass spectrometry, and chemical synthesis, the structure of this unusual GSL was established to be a taurine-conjugated GM2 (tauro-GM2) in which the carboxyl group of N-acetylneuraminic acid was amidated by taurine. Using a rabbit anti-tauro-GM2 serum, we also detected the presence of tauro-GM2 in three other small brain samples from one variant B and two variant O TSD patients. On the other hand, tauro-GM2 was not found in three normal human brain samples. The presence of tauro-GM2 in TS brains, but not in normal brains, indicates the possible association of this unusual GM2 derivative with the pathogenesis of TSD. Our findings point to taurine conjugation as a heretofore unelucidated mechanism for TS brain to cope with water-insoluble GM2.

Fragmentation pathway studies of oligonucleotides in matrix-assisted laser desorption/ionization mass spectrometry by charge tagging and H/D exchange.

The desorption and decompositions of synthesized oligonucleotides bearing fixed charge sites have been investigated by linear, delayed-extraction, reflecting and post-source decay mode matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. In contrast to the conventional [M + H]+ forms of unmodified molecules where a proton is likely attached to a nucleobase, here the charge is fixed at one of the termini. In this case the observed fragment ions always incorporate the charge-tag. H/D exchange experiments provide no evidence for intramolecular migration of protons on the phosphate backbone to initiate the fragmentation event. New unique pathways of proton migration from the ribose have been elucidated and are rationalized by a charge-remote fragmentation pathway.