Salinity as a regulator of DMSP degradation in Ruegeria pomeroyi DSS-3

Dimethylsulfoniopropionate (DMSP) is an important carbon and sulfur source to marine bacterial communities and the main precursor of dimethylsulfide (DMS), a gas that influences atmospheric chemistry and potentially the global climate. In nature, bacterial DMSP catabolism can yield different proportions of DMS and methanethiol (MeSH), but relatively little is known about the factors controlling the pathways of bacterial degradation that select between their formation (cleavage vs. demethiolation). In this study, we carried out experiments to evaluate the influence of salinity on the routes of DMSP catabolism in Ruegeria pomeroyi DSS-3. We monitored DMS and MeSH accumulation in cell suspensions grown in a range of salinities (10, 20, 30 ppt) and with different DMSP amendments (0, 50, 500 µM). Significantly higher concentrations of DMS accumulated in low salinity treatments (10 ppt; P < 0.001), in both Marine Basal Medium (MBM) and half-strength Yeast Tryptone Sea Salts (1/2 YTSS) media. Results showed a 47.1% and 87.5% decrease of DMS accumulation, from salinity 10 to 20 ppt, in MBM and 1/2 YTSS media, respectively. On the other hand, MeSH showed enhanced accumulations at higher salinities (20, 30 ppt), with a 90.6% increase of MeSH accumulation from the 20 ppt to the 30 ppt salinity treatments. Our results with R. pomeroyi DSS-3 in culture are in agreement with previous results from estuarine sediments and demonstrate that salinity can modulate selection of the DMSP enzymatic degradation routes, with a consequent potential impact on DMS and MeSH liberation into the atmosphere.     

Evidence That the Pathway of Dimethylsulfoniopropionate Biosynthesis Begins in the Cytosol and Ends in the Chloroplast

In the flowering plant Wollastonia biflora (L.) DC. the first step in 3-dimethylsulfoniopropionate (DMSP) synthesis is conversion of methionine to S-methylmethionine (SMM) and the last is oxidation of 3-dimethylsulfoniopropionaldehyde (DMSP-ald) (F. James, L. Paquet, S.A. Sparace, D.A. Gage, A.D. Hanson [1995] Plant Physiol 108: 1439-1448). DMSP-ald was shown to undergo rapid, spontaneous decomposition to dimethylsulfide and acrolein. However, it was stable enough (half-life [greater than or equal to] 1 h) in tertiary amine buffers to use as a substrate for enzyme assays. A dehydrogenase catalyzing DMSP-ald oxidation was detected in extracts of W. biflora mesophyll protoplasts. This enzyme had a high affinity for DMSP-ald (Km = 1.5 [mu]M), was subject to substrate inhibition, preferred NAD to NADP, and was immunologically related to plant betaine aldehyde dehydrogenases. After fractionation of protoplast lysates, [greater than or equal to]90% of DMSP-ald dehydrogenase activity was recovered from the chloroplast stromal fraction, whereas the enzyme that mediates SMM synthesis, S-adenosylmethionine:methionine S-methyltransferase, was found exclusively in the cytosolic fraction. Immunohistochemical analysis confirmed that the S-methyltransferase was cytosolic. Intact W. biflora chloroplasts were able to metabolize supplied [35S]SMM to [35S]DMSP. These findings indicate that SMM is made in the cytosol, imported into the chloroplast, and there converted successively to DMSP-ald and DMSP.     

Overexpressing 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase (HMGR) in the Lactococcal Mevalonate Pathway for Heterologous Plant Sesquiterpene Production

Isoprenoids are a large and diverse group of metabolites with interesting properties such as flavour, fragrance and therapeutic properties. They are produced via two pathways, the mevalonate pathway or the 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway. While plants are the richest source of isoprenoids, they are not the most efficient producers. Escherichia coli and yeasts have been extensively studied as heterologous hosts for plant isoprenoids production. In the current study, we describe the usage of the food grade Lactococcus lactis as a potential heterologous host for the production of sesquiterpenes from a local herbaceous Malaysian plant, Persicaria minor (synonym Polygonum minus). A sesquiterpene synthase gene from P. minor was successfully cloned and expressed in L. lactis. The expressed protein was identified to be a β-sesquiphellandrene synthase as it was demonstrated to be functional in producing β-sesquiphellandrene at 85.4% of the total sesquiterpenes produced based on in vitro enzymatic assays. The recombinant L. lactis strain developed in this study was also capable of producing β-sesquiphellandrene in vivo without exogenous substrates supplementation. In addition, overexpression of the strain’s endogenous 3-hydroxy-3-methylglutaryl coenzyme-A reductase (HMGR), an established rate-limiting enzyme in the eukaryotic mevalonate pathway, increased the production level of β-sesquiphellandrene by 1.25–1.60 fold. The highest amount achieved was 33 nM at 2 h post-induction.     

The E3 Ubiquitin Ligase MARCH6 Degrades Squalene Monooxygenase and Affects 3-Hydroxy-3-Methyl-Glutaryl Coenzyme A Reductase and the Cholesterol Synthesis Pathway

The mevalonate pathway is used by cells to produce sterol and nonsterol metabolites and is subject to tight metabolic regulation. We recently reported that squalene monooxygenase (SM), an enzyme controlling a rate-limiting step in cholesterol biosynthesis, is subject to cholesterol-dependent proteasomal degradation. However, the E3-ubiquitin (E3) ligase mediating this effect was not established. Using a candidate approach, we identify the E3 ligase membrane-associated RING finger 6 (MARCH6, also known as TEB4) as the ligase controlling degradation of SM. We find that MARCH6 and SM physically interact, and consistent with MARCH6 acting as an E3 ligase, its overexpression reduces SM abundance in a RING-dependent manner. Reciprocally, knockdown of MARCH6 increases the level of SM protein and prevents its cholesterol-regulated degradation. Additionally, this increases cell-associated SM activity but is unexpectedly accompanied by increased flux upstream of SM. Prompted by this observation, we found that knockdown of MARCH6 also controls the level of 3-hydroxy-3-methyl-glutaryl coenzyme A reductase (HMGCR) in hepatocytes and model cell lines. In conclusion, MARCH6 controls abundance of both SM and HMGCR, establishing it as a major regulator of flux through the cholesterol synthesis pathway.     

Functional Analysis of Glycosyltransferase Genes from Lactococcus lactis and Other Gram-Positive Cocci: Complementation, Expression, and Diversity

Sixteen exopolysaccharide (EPS)-producing Lactococcus lactis strains were analyzed for the chemical compositions of their EPSs and the locations, sequences, and organization of the eps genes involved in EPS biosynthesis. This allowed the grouping of these strains into three major groups, representatives of which were studied in detail. Previously, we have characterized the eps gene cluster of strain NIZO B40 (group I) and determined the function of three of its glycosyltransferase (GTF) genes. Fragments of the eps gene clusters of strains NIZO B35 (group II) and NIZO B891 (group III) were cloned, and these encoded the NIZO B35 priming galactosyltransferase, the NIZO B891 priming glucosyltransferase, and the NIZO B891 galactosyltransferase involved in the second step of repeating-unit synthesis. The NIZO B40 priming glucosyltransferase gene epsD was replaced with an erythromycin resistance gene, and this resulted in loss of EPS production. This epsD deletion was complemented with priming GTF genes from gram-positive organisms with known function and substrate specificity. Although no EPS production was found with priming galactosyltransferase genes from L. lactis or Streptococcus thermophilus, complementation with priming glucosyltransferase genes involved in L. lactis EPS and Streptococcus pneumoniae capsule biosynthesis could completely restore or even increase EPS production in L. lactis.     

Comparative Analysis of Transmembrane Regulators of the Filamentous Growth Mitogen-Activated Protein Kinase Pathway Uncovers Functional and Regulatory Differences

Filamentous growth is a microbial differentiation response that involves the concerted action of multiple signaling pathways. In budding yeast, one pathway that regulates filamentous growth is a Cdc42p-dependent mitogen-activated protein kinase (MAPK) pathway. Several transmembrane (TM) proteins regulate the filamentous growth pathway, including the signaling mucin Msb2p, the tetraspan osmosensor Sho1p, and an adaptor Opy2p. The TM proteins were compared to identify common and unique features. Msb2p, Sho1p, and Opy2p associated by coimmunoprecipitation analysis but showed predominantly different localization patterns. The different localization patterns of the proteins resulted in part from different rates of turnover from the plasma membrane (PM). In particular, Msb2p (and Opy2p) were turned over rapidly compared to Sho1p. Msb2p signaled from the PM, and its turnover was a rate-limiting step in MAPK signaling. Genetic analysis identified unique phenotypes of cells overexpressing the TM proteins. Therefore, each TM regulator of the filamentous growth pathway has its own regulatory pattern and specific function in regulating filamentous growth. This specialization may be important for fine-tuning and potentially diversifying the filamentation response.     

Formyltetrahydrofolate Synthetase Sequences from Salt Marsh Plant Roots Reveal a Diversity of Acetogenic Bacteria and Other Bacterial Functional Groups

Sixty-two partial formyltetrahydrofolate synthetase (FTHFS) structural gene sequences were recovered from roots of salt marsh plants, including Spartina alterniflora, Salicornia virginica, and Juncus roemerianus. Only S. alterniflora roots yielded sequences grouping with FTHFS sequences from known acetogens. Most other FTHFS or FTHFS-like sequences grouped with those from sulfate-reducing bacteria. Several sequences that grouped with Sphingomonas paucimobilis ligH were also recovered.     

Functional Pathway Analysis Using SCNP of FLT3 Receptor Pathway Deregulation in AML Provides Prognostic Information Independent from Mutational Status

FMS-like tyrosine kinase 3 receptor (FLT3) internal tandem duplication (ITD) mutations result in constitutive activation of this receptor and have been shown to increase the risk of relapse in patients with acute myeloid leukemia (AML); however, substantial heterogeneity in clinical outcomes still exists within both the ITD mutated and unmutated AML subgroups, suggesting alternative mechanisms of disease relapse not accounted by FLT3 mutational status. Single cell network profiling (SCNP) is a multiparametric flow cytometry based assay that simultaneously measures, in a quantitative fashion and at the single cell level, both extracellular surface marker levels and changes in intracellular signaling proteins in response to extracellular modulators. We previously reported an initial characterization of FLT3 ITD-mediated signaling using SCNP. Herein SCNP was applied sequentially to two separate cohorts of samples collected from elderly AML patients at diagnosis. In the first (training) study, AML samples carrying unmutated, wild-type FLT3 (FLT3 WT) displayed a wide range of induced signaling, with a fraction having signaling profiles comparable to FLT3 ITD AML samples. Conversely, the FLT3 ITD AML samples displayed more homogeneous induced signaling, with the exception of patients with low (<40%) mutational load, which had profiles comparable to FLT3 WT AML samples. This observation was then confirmed in an independent (verification) cohort. Data from the second cohort were also used to assess the association between SCNP data and disease-free survival (DFS) in the context of FLT3 and nucleophosmin (NPM1) mutational status among patients who achieved complete remission (CR) to induction chemotherapy. The combination of SCNP read outs together with FLT3 and NPM1 molecular status improved the DFS prediction accuracy of the latter. Taken together, these results emphasize the value of comprehensive functional assessment of biologically relevant signaling pathways in AML as a basis for the development of highly predictive tests for guidance of post-remission therapy.     

c-Cbl and Cbl-b Ligases Mediate 17-Allylaminodemethoxygeldanamycin-induced Degradation of Autophosphorylated Flt3 Kinase with Internal Tandem Duplication through the Ubiquitin Proteasome Pathway

The class III receptor-tyrosine kinase Flt3 regulates normal hematopoiesis. An internal tandem duplication (ITD) in the juxtamembrane domain of Flt3 (Flt3-ITD) contributes to transformation and is associated with poor prognosis in acute myeloid leukemia. Here, we demonstrate that, as compared with wild-type Flt3 (Flt3-WT), Flt3-ITD more rapidly undergoes degradation through the proteasomal and lysosomal pathways in model hematopoietic 32D cells and in human leukemic MV4-11 cells. The Hsp90 inhibitor 17-allylaminodemethoxygeldanamycin (17-AAG) preferentially induced the polyubiquitination and proteasomal degradation of Flt3-ITD autophosphorylated on Tyr-591 in these cells. The E3 ubiquitin ligases c-Cbl and to a lesser extent Cbl-b facilitated at least partly Lys-48-linked polyubiquitination of autophosphorylated Flt3-ITD when coexpressed in 293T cells. Moreover, c-Cbl and Cbl-b facilitated degradation of Flt3-ITD in 293T cells and significantly enhanced the 17-AAG-induced decline in autophosphorylated Flt3-ITD. The enhancement of Flt3-ITD degradation was also observed in 32D cells inducibly overexpressing c-Cbl or Cbl-b. Furthermore, overexpression of loss-of-function mutants of both c-Cbl (c-Cbl-R420Q) and Cbl-b (Cbl-b-C373A) together in 32D cells retarded the degradation of autophosphorylated Flt3-ITD and significantly inhibited the 17-AAG-induced degradation of Flt3-ITD to confer the resistance to cytotoxicity of 17-AAG on these cells. These results suggest that c-Cbl as well as Cbl-b may play important roles in Hsp90 inhibitor-induced degradation of Flt3-ITD through the ubiquitin proteasome system and in regulation of the basal expression level of Flt3-ITD in leukemic cells.     

Genetic Diversity of Benzoyl Coenzyme A Reductase Genes Detected in Denitrifying Isolates and Estuarine Sediment Communities

Benzoyl coenzyme A (benzoyl-CoA) reductase is a central enzyme in the anaerobic degradation of organic carbon, which utilizes a common intermediate (benzoyl-CoA) in the metabolism of many aromatic compounds. The diversity of benzoyl-CoA reductase genes in denitrifying bacterial isolates capable of degrading aromatic compounds and in river and estuarine sediment samples from the Arthur Kill in New Jersey and the Chesapeake Bay in Maryland was investigated. Degenerate primers were developed from the known benzoyl-CoA reductase genes from Thauera aromatica, Rhodopseudomonas palustris, and Azoarcus evansii. PCR amplification detected benzoyl-CoA reductase genes in the denitrifying isolates belonging to α-, β-, or γ-Proteobacteria as well as in the sediment samples. Phylogenetic analysis, sequence similarity comparison, and conserved indel determination grouped the new sequences into either the bcr type (found in T. aromatica and R. palustris) or the bzd type (found in A. evansii). All the Thauera strains and the isolates from the genera Acidovorax, Bradyrhizobium, Paracoccus, Ensifer, and Pseudomonas had bcr-type benzoyl-CoA reductases with amino acid sequence similarities of more than 97%. The genes detected from Azarocus strains were assigned to the bzd type. A total of 50 environmental clones were detected from denitrifying consortium and sediment samples, and 28 clones were assigned to either the bcr or the bzd type of benzoyl-CoA reductase genes. Thus, we could determine the genetic capabilities for anaerobic degradation of aromatic compounds in sediment communities of the Chesapeake Bay and the Arthur Kill on the basis of the detection of two types of benzoyl-CoA reductase genes. The detected genes have future applications as genetic markers to monitor aromatic compound degradation in natural and engineered ecosystems.     

Isolation and Characterization of Mutants of Clostridium acetobutylicum ATCC 824 Deficient in Acetoacetyl-Coenzyme A:Acetate/Butyrate:Coenzyme A-Transferase (EC 2.8.3.9) and in Other Solvent Pathway Enzymes

Mutants of Clostridium acetobutylicum ATCC 824 exhibiting resistance to 2-bromobutyrate or rifampin were isolated after nitrosoguanidine treatment. Mutants were screened for solvent production by using an automated alcohol test system. Isolates were analyzed for levels of butanol, ethanol, acetone, butyrate, acetate, and acetoin in stationary-phase batch cultures. The specific activities of NADH- and NADPH-dependent butanol dehydrogenase and butyraldehyde dehydrogenase as well as those of acetoacetyl-coenzyme A:acetate/butyrate:coenzyme A-transferase (butyrate-acetoacetate coenzyme A-transferase [EC 2.8.3.9]) (CoA-transferase), butyrate kinase, and phosphotransbutyrylase were measured at the onset of stationary phase. Rifampin-resistant strain D10 and 2-bromobutyrate mutant R were found to be deficient in only CoA-transferase, while several other mutants exhibited reduced butyraldehyde dehydrogenase and butanol dehydrogenase activities as well. The colony morphology of 2-bromobutyrate mutant R was similar to that of the parent on RCM medium; however, it had about 1/10 the level of CoA-transferase and increased levels of butanol dehydrogenase and butyraldehyde dehydrogenase. A nonsporulating, spontaneously derived degenerated strain exhibited reduced levels of butyraldehyde dehydrogenase, butanol, dehydrogenase, and CoA-transferase compared with those of the original strain. When C. acetobutylicum ATCC 824 was grown on medium containing low levels of 2-bromobutyrate, an altered colony morphology was observed. Not all strains resistant to 2-bromobutyrate (12 mM) were non-solvent-producing strains.     

Regulatory Roles of the PI3K/Akt Signaling Pathway in Rats with Severe Acute Pancreatitis

The phosphatidylinositol 3-kinase(PI3K)/protein kinase B (Akt) pathway plays a key role in inflammation. However, the regulatory roles of PI3K/Akt in severe acute pancreatitis (SAP) have not been elucidated. The aim of this study was to investigate the impact of wortmannin, a PI3K/Akt inhibitor, on SAP rats through exposure to sodium taurocholate (STC) after 3 h and 6 h. The SAP group was found to have a significant increase in pancreas Akt expression, along with the activation of serum amylase, TNF-α, IL-1β, and IL-6, and pancreas histological aggravation. The administration of wortmannin in SAP rats reduced Akt expression, attenuated the level of serum amylase and inflammation factor, and alleviated the damage of pancreatic tissue. Furthermore, the administration of wortmannin led to an obvious reduction in NF-κB and p38MAPK expression in SAP rats. These findings showed that the PI3K/Akt inhibitor wortmannin decreases inflammatory cytokines in SAP rats and suggests its regulatory mechanisms may occur through the suppression on NF-κB and p38MAPK activity.     

A Proteomic Approach To Identify Candidate Substrates of Human Adenovirus E4orf6-E1B55K and Other Viral Cullin-Based E3 Ubiquitin Ligases

It has been known for some time that the human adenovirus serotype 5 (Ad5) E4orf6 and E1B55K proteins work in concert to degrade p53 and to regulate selective export of late viral mRNAs during productive infection. Both of these functions rely on the formation by the Ad5 E4orf6 protein of a cullin 5-based E3 ubiquitin ligase complex containing elongins B and C. E1B55K is believed to function as the substrate recognition module for the complex and, in addition to p53, Mre11 and DNA ligase IV have also been identified as substrates. To discover additional substrates we have taken a proteomic approach by using two-dimensional difference gel electrophoresis to detect cellular proteins that decrease significantly in amount in p53-null H1299 human lung carcinoma cells after expression of E1B55K and E4orf6 using adenovirus vectors. Several species were detected and identified by mass spectroscopy, and for one of these, integrin α3, we went on in a parallel study to confirm it as a bone fide substrate of the complex (F. Dallaire et al., J. Virol. 83:5329-5338, 2009). Although the system has some limitations, it may still be of some general use in identifying candidate substrates of any viral cullin-based E3 ubiquitin ligase complex, and we suggest a series of criteria for substrate validation.During the past decade protein degradation has become increasingly recognized as a critical mechanism by which cells regulate a number of fundamental processes (reviewed in references 37, 57, and 59). Degradation frequently involves one of a variety of E3 ubiquitin ligase complexes in which a substrate recognition component introduces the target protein for ubiquitination and subsequent degradation by proteasomes (reviewed in reference 59). Several types of these complexes involve a member of the cullin family (reviewed in reference 59), and a considerable amount of information is known about those containing Cul2 or Cul5. In these cases the substrate recognition module is linked via elongins B and C to a subcomplex containing Cul2 or Cul5 and the RING protein Rbx1 (34, 58). This complex interacts with an E2 conjugating enzyme, often either Cdc34 or Ubc5, to conjugate ubiquitin chains to the substrate (44). With both Cul2- and Cul5-based complexes interaction with elongins B and C occurs via a single BC box sequence (42). The presence of either Cul2 or Cul5 is generally determined through the presence in the substrate recognition protein of specific Cul2- or Cul5-box sequences (35).Many viruses have evolved to encode products that inhibit cellular E3 ligases to protect important viral or cellular species or, in some cases, that highjack these cellular complexes to target key substrates for degradation, including components of cellular host defenses, to facilitate the infectious cycle (reviewed in reference 4). These strategies are quite common among the small DNA tumor viruses (7), and one of the most studied examples is the complex formed by the human adenovirus E4orf6 and E1B55K proteins. These proteins have been known for some time to interact (69) and to reduce the levels of the p53 tumor suppressor in infected cells (14, 47, 48, 62, 72, 73). In addition, they were shown to function in concert to block nuclear export of cellular mRNAs late in infection (2, 6, 29, 60) and to enhance the selective export of late viral mRNAs (2, 26, 29, 60, 78). Our group showed that the human adenovirus serotype 5 (Ad5) E4orf6 product interacts with several proteins (13), including components of what was at the time a unique Cul5-based E3 ubiquitin ligase containing elongins B and C and Rbx1 that degrades p53 (61). Curiously, Ad5 E4orf6 contains three BC boxes that we believe make it highly efficient in highjacking cellular elongin B/C complexes (8, 17, 41). The mechanism of selective recruitment of Cul5 by the Ad5 complex remains unknown as E4orf6 lacks a Cul5-box (17, 41). E1B55K seems to function as the substrate recognition module and, of considerable interest, both its association with E4orf6 and induction of selective late viral mRNA transport was found to depend on formation of the E3 ubiquitin ligase complex, suggesting that additional degradation substrates must exist (8, 9). This idea is not surprising since viruses, especially the small DNA tumor viruses, often evolve gene products that target multiple critical cellular pathways (32). In fact two additional E1B55K-binding substrates have now been identified, Mre11 from the MRN DNA repair complex (8, 75), and DNA ligase IV (3), the degradation of which prevent formation of viral genome concatemers, thus enhancing packaging of progeny DNA. Degradation of p53 has been suggested to promote enhanced progeny virus production by preventing the early apoptotic death of infected cells due to the stabilization of p53 by the viral E1A products (reviewed in reference 66). Nevertheless, degradation of these substrates seems unlikely to explain the observed effects on mRNA transport, suggesting that still more substrates remain to be identified. Although the studies described in the present report were in part launched to identify such substrates, as will become clear below, these targets remain to be identified.In an attempt to identify new substrates of the Ad5 E4orf6/E1B55K E3 ubiquitin ligase complex, a proteomics-based approach was initiated involving two-dimensional difference gel electrophoresis (2D-DIGE) analysis and subsequent mass spectrometry. As is well known, this technique has the advantage of improved sensitivity and accuracy provided by its ability to separate samples under two different conditions on a single gel together with a reference sample, thus reducing significantly the analytical coefficient of variation. It allows the quantification of differentially abundant proteins in complex biological samples, providing a tool to detect decreases in the levels of proteins in the cell due to targeted proteolytic degradation. We report here our attempts to identify substrates of the Ad5 E4orf6/E1B55K complex by comparing the proteomes of human non-small cell lung carcinoma H1299 cells expressing, by means of adenovirus vectors, both E1B55K and E4orf6 proteins or E4orf6 protein alone. Ten candidate proteins were identified, most having functions seemingly unrelated to our current understanding of the roles of the E4orf6/E1B55K complex. At least three showed promising features characteristic of substrates, and one has now been confirmed in a parallel study to be a bone fide E4orf6/E1B55K substrate (20). We suggest that this approach could be utilized to identify candidate substrates, among relatively high abundance proteins, that are degraded by other viral cullin-based E3 ubiquitin ligase complexes.