Chaetomella acutiseta produces chaetomellic acids A and B which are reversible inhibitors of farnesyl-protien transferase

Chaetomellic acids A and B, isolated from Chaetomella acutiseta, are specific inhibitors of farnesyl-protein transferase that do not inhibit geranylgeranyl transferase type 1 or squalene synthase. Chaetomellic acids A and B are reversible inhibitors, resemble farnesyl diphosphate and probably inhibit FPTase by substituting for farnesyl diphosphate. Chaetomellic acid production appears to be widespread within the genus Chaetomella. Correspondence to: R. B. Lingham     

High-Level Expression of Rat Farnesyl:Protein Transferase inEscherichia colias a Translationally Coupled Heterodimer

Farnesyl:protein transferase (FPTase) catalyzes the transfer of a 15-carbon farnesyl isoprenoid group from farnesyl diphosphate to the CaaX cysteine of a variety of cellular proteins. Since FPTase is a large (95-kDa) heterodimeric protein and is inactive unless the α- and β-subunits are coexpressed, large-scale overexpression of active enzyme has been challenging. We report the design of a translationally coupled expression system that will produce FPTase at levels as high as 30 mg/LEscherichia coli.Heterodimeric expression of FPTase was achieved using a translationally coupled operon from the T7 promoter of the pET23a (Novagen) expression plasmid. The β-subunit-coding sequence was placed upstream of the α-subunit coding sequence linked by overlapping β-subunit stop and α-subunit start codons. Additionally, the initial 88 codons of the α-subunit gene were altered, removing rare codons and replacing them with codons used in highly expressed proteins inE. coli.Since previous attempts at recombinantly expressing FPTase inE. colifrom a translationally coupled system have demonstrated that initiation of translation of the α-subunit is poor, we propose that the optimization of the codons at the start of the α-subunit gene leads to the observed high level of recombinant expression.     

Polysaccharide-specific probes inhibit adhesion of Hyphomonas rosenbergii strain VP-6 to hydrophilic surfaces

Biofilm formation commences with the adhesion of microorganisms to surfaces. Information regarding the initial bond between a bacterium and a solid surface is essential for devising methods to inhibit the onset of biofilm formation. Three different types of polysaccharide-specific probes, cationic metals, dyes, and lectins, were used to bind the exopolysaccharide of Hyphomonas rosenbergii, a budding, prosthecate marine bacterium. Probes, which specifically bind complex carbohydrates, inhibit the adhesion of H. rosenbergii to hydrophilic surfaces. These results suggest that the polysaccharide portion of H. rosenbergii capsular, extracellular polymeric-substance is involved in the primary adhesion process. Journal of Industrial Microbiology & Biotechnology (2000) 25, 81–85. Received 01 February 2000/ Accepted in revised form 03 June 2000     

A theoretical approach to spot active regions in antimicrobial proteins

Background  

Much effort goes into identifying new antimicrobial compounds able to evade the increasing resistance of microorganisms to antibiotics. One strategy relies on antimicrobial peptides, either derived from fragments released by proteolytic cleavage of proteins or designed from known antimicrobial protein regions.     

Synthesis and evaluation of a novel series of farnesyl protein transferase inhibitors as non-peptidic CAAX tetrapeptide analogues

A novel series of compounds, derived from 4-amino-phenyl piperazine, has been designed to selectively inhibit farnesyl protein transferase (FPTase) as CAAX tetrapeptide analogues. Certain of these compounds were shown to possess low nanomolar inhibitory activity both against the isolated enzyme and in cultured cells.     

A novel <Emphasis Type="Italic">Dictyostelium</Emphasis> RasGEF required for chemotaxis and development

Background  

Ras proteins are guanine-nucleotide-binding enzymes that couple cell surface receptors to intracellular signaling pathways controlling cell proliferation and differentiation, both in lower and higher eukaryotes. They act as molecular switches by cycling between active GTP and inactive GDP-bound states, through the action of two classes of regulatory proteins: a) guanine nucleotide exchange factor (GEFs) and b) GTP-ase activating proteins (GAPs). Genome wide analysis of the lower eukaryote Dictyostelium discoideum revealed a surprisingly large number of Ras Guanine Nucleotide Exchange Factors (RasGEFs). RasGEFs promote the activation of Ras proteins by catalyzing the exchange of GDP for GTP, thus conferring to RasGEFs the role of main activator of Ras proteins. Up to date only four RasGEFs, which are all non-redundant either for growth or development, have been characterized in Dictyostelium. We report here the identification and characterization of a fifth non-redundant GEF, RasGEFM.     

Spatiotemporal Organization of Ras Signaling: Rasosomes and the Galectin Switch

Summary 1. Ras signaling and oncogenesis depend on the dynamic interplay of Ras with distinctive plasma membrane (PM) microdomains and various intracellular compartments. Such interaction is dictated by individual elements in the carboxy-terminal domain of the Ras proteins, including a farnesyl isoprenoid group, sequences in the hypervariable region (hvr)-linker, and palmitoyl groups in H/N-Ras isoforms.2. The farnesyl group acts as a specific recognition unit that interacts with prenyl-binding pockets in galectin-1 (Gal-1), galectin-3 (Gal-3), and cGMP phosphodiesterase δ. This interaction appears to contribute to the prolongation of Ras signals in the PM, the determination of Ras effector usage, and perhaps also the transport of cytoplasmic Ras. Gal-1 promotes H-Ras signaling to Raf at the expense of phosphoinositide 3-kinase (PI3-K) and Ral guanine nucleotide exchange factor (RalGEF), while galectin-3 promotes K-Ras signaling to both Raf and PI3-K.3. The hvr-linker and the palmitates of H-Ras and N-Ras determine the micro- and macro-localizations of these proteins in the PM and in the Golgi, as well as in ‘rasosomes’, randomly moving nanoparticles that carry palmitoylated Ras proteins and their signal through the cytoplasm.4. The dynamic compartmentalization of Ras proteins contributes to the spatial organization of Ras signaling, promotes redistribution of Ras, and provides an additional level of selectivity to the signal output of this regulatory GTPase.     

Protein prenylation in eukaryotic microorganisms: genetics, biology and biochemistry

Modrfication of proteins at C-terminal cysteine residue(s) by the isoprenoids farnesyl (C15) and geranylgeranyl (C20) is essential for the biological function of a number of eukaryotic proteins including fungal mating factors and the small, GTP-binding proteins of the Ras superfamily. Three distinct enzymes, conserved between yeast and mammals, have been identified that prenylate proteins: farnesyl protein transferase, geranylgeranyl protein transferase type I and geranylgeranyl protein transferase type II. Each prenyl protein transferase has its own protein substrate specificity. Much has been learned about the biology, genetics and biochemistry of protein prenylation and prenyl protein transferases through studies of eukaryotic microorganisms, particularly Saccharo-myces cerevisiae. The functional Importance of protein prenylation was first demonstrated with fungal mating factors. The initial genetic analysis of prenyl protein transferases was in S. cerewisiae with the isolation and subsequent characterization of mutations in the RAM1, RAM2, CDC43 and BET2 genes, each of which encodes a prenyl protein transferase subunit. We review here these and other studies on protein prenylation in eukaryotic microbes and how they relate to and have contributed to our knowledge about protein prenylation in all eukaryotic cells.     

Prevention of farnesylation of c-Ha-Ras protein enhances synergistically the cytotoxic action of doxorubicin in cycling but not in quiescent cells

Ras, the product of a proto-oncogene, is a GTP-hydrolyzing enzyme found mutated in approximately 50% of human cancers. "Gain of function" mutations of Ras lead to an escape of transformed cells from cell-cycle control, rendering them independent to stimulation by growth factors, giving them almost unlimited proliferation capacity. The cytosolic precursor isoform of Ras is biologically inactive. After several post-translational modifications, Ras is anchored to the plasma membrane and, thereby, the protein becomes activated. The finding that lipid modifications of Ras protein, particularly farnesylation, are essential for its signal transduction activity, gave rise to the concept that blocking farnesyl protein transferase (FPTase), the enzyme catalyzing the first step in the Ras modification cascade, would prevent proper membrane anchoring and provide an improved approach in the cure of tumors harboring Ras mutations. In the present study we used transformed rat cells overexpressing a temperature-sensitive p53 protein, adopting wt conformation at 32 degrees C and mutant conformation at 37 degrees C. We treated the cells growing at 32 or 37 degrees C with doxorubicin alone, or in combination with inhibitors of FPTase. Combined treatment was more efficient and the same inhibition of cell proliferation was reached at lower DOX concentrations. The treatment strongly affected the growth rate of tumor cells but only negligibly of normal cells. However, the inhibitors of FPTase prevented the membrane anchoring in both situations. These results show two striking advantages of the combined treatment: the desired cytostatic effect on tumor cells at lower drug concentrations and clearly reduced adverse effects on quiescent cells.     

The synthesis and biological evaluation of a series of potent dual inhibitors of farnesyl and geranyl-Geranyl protein transferases

We have prepared a series of potent, dual inhibitors of the prenyl transferases farnesyl protein transferase (FPTase) and geranyl-geranyl protein transferase I (GGPTase). The compounds were shown to possess potent activity against both enzymes in cell culture. Mechanistic analysis has shown that the compounds are CAAX competitive for FPTase inhibition but geranyl-geranyl pyrophosphate (GGPP) competitive for GGPTase inhibiton.     

Leishmania donovani infection differentially regulates small G‐proteins

Leishmania is a protozoan parasite that resides and replicates in macrophages and causes leishmaniasis. The parasite alters the signaling cascade in host macrophages and evades the host machinery. Small G‐proteins are GTPases, grouped in 5 different families that play a crucial role in the regulation of cell proliferation, cell survival, apoptosis, intracellular trafficking, and transport. In particular, the Ras family of small G‐proteins has been identified to play a significant role in the cellular functions mentioned before. Here, we studied the differential expression of the most important small G‐proteins during Leishmania infection. We found major changes in the expression of different isoforms of Ras, mainly in N‐Ras. We observed that Leishmania donovani infection led to enhanced N‐Ras expression, whereas it inhibited K‐Ras and H‐Ras expression. Furthermore, an active N‐Ras pull‐down assay showed enhanced N‐Ras activity. L donovani infection also increased extracellular signal–regulated kinase 1/2 phosphorylation and simultaneously decreased p38 phosphorylation. In contrast, pharmacological inhibition of Ras led to reduction in the phosphorylation of extracellular signal–regulated kinase 1/2 and enhanced the phosphorylation of p38 in Leishmania‐infected cells, which could lead to increased interleukin‐12 expression and decreased interleukin‐10 expression. Indeed, farnesylthiosalicyclic acid (a Ras inhibitor), when used at the effective level in L donovani–infected macrophages, reduced amastigotes in the host macrophages. Thus, upregulated N‐Ras expression during L donovani infection could be a novel immune evasion strategy of Leishmania and would be a potential target for antileishmanial immunotherapy.     

Synthesis and preliminary pharmacological characterisation of a new class of nitrogen-containing bisphosphonates (N-BPs)

A new series of bisphosphonates bearing either the nitrogen-containing NO-donor furoxan (1,2,5-oxadiazole 2-oxide) system or the related furazan (1,2,5-oxadiazole) in lateral chain has been developed. pK_a values and affinity for hydroxyapatite were determined for all the compounds. The products were able to inhibit osteoclastogenesis on RAW 246.7 cells at 10 μM concentration. The most active compounds were further assayed on human PBMC cells and on rat microsomes. Unlike most nitrogen-containing bisphosphonates which target farnesyl pyrophosphate synthase, experimental and theoretical investigations suggest that the activity of our derivatives may be related to different mechanisms. The furoxan derivatives were also tested for their ability to relax rat aorta strips in view of their potential NO-dependent vasodilator properties.     

SH3‐class Ras guanine nucleotide exchange factors are essential for Aspergillus fumigatus invasive growth

Proper hyphal morphogenesis is essential for the establishment and progression of invasive disease caused by filamentous fungi. In the human pathogen Aspergillus fumigatus, signalling cascades driven by Ras and Ras‐like proteins orchestrate a wide variety of cellular processes required for hyphal growth. For activation, these proteins require interactions with Ras‐subfamily‐specific guanine nucleotide exchange factors (RasGEFs). Although Ras‐protein networks are essential for virulence in all pathogenic fungi, the importance of RasGEF proteins is largely unexplored. A. fumigatus encodes four putative RasGEFs that represent three separate classes of RasGEF proteins (SH3‐, Ras guanyl nucleotide‐releasing protein [RasGRP]–, and LTE‐class), each with fungus‐specific attributes. Here, we show that the SH3‐class and RasGRP‐class RasGEFs are required for properly timed polarity establishment during early growth and branch emergence as well as for cell wall stability. Further, we show that SH3‐class RasGEF activity is essential for polarity establishment and maintenance, a phenotype that is, at least, partially independent of the major A. fumigatus Ras proteins, RasA and RasB. Finally, loss of both SH3‐class RasGEFs resulted in avirulence in multiple models of invasive aspergillosis. Together, our findings suggest that RasGEF activity is essential for the integration of multiple signalling networks to drive invasive growth in A. fumigatus.     

Conformation of a novel tetrapeptide inhibitor NH2-D-Trp-D-Met-Phe(pCl)-Gla-NH2 bound to farnesyl-protein transferase.

Farnesyl-protein transferase (FPTase) catalyzes the posttranslational farnesylation of the cysteine residue located in the C-terminal tetrapeptide of the Ras oncoprotein. Prenylation of this residue is essential for membrane association and cell-transforming activities of ras. Inhibitors of FPTase have been demonstrated to display antitumor activity in both tissue culture and animal models, and thus represent a potential therapeutic strategy for the treatment of human cancers. A synthetic tetrapeptide library, which included an expanded set of 68 L-, D- and noncoded amino acids, has been screened for inhibitors of FPTase activity. The tetrapeptide, NH2-D-Trp-D-Met-L-Phe(pCl)-L-Gla-NH2 was shown to be competitive with the isoprenyl cosubstrate, farnesyl diphosphate (FPP) but not with the peptide substrate, the C-terminal tetrapeptide of the Ras protein. The FPTase-bound conformation of the inhibitor, NH2-D-Trp-D-Met-L-Phe(pCl)-L-Gla-NH2 was determined by NMR spectroscopy. Distance constraints were derived from two-dimensional transferred nuclear Overhauser effect (TRNOE) experiments. Ligand competition experiments identified the NOEs that originate from the active-site conformation of the inhibitor. Structures were calculated using a combination of distance geometry and restrained energy minimization. The peptide backbone is shown to adopt a reverse-turn conformation most closely approximating a type II' beta-turn. The resolved conformation of the inhibitor represents a distinctly different structural motif from that determined for Ras-competitive inhibitors. Knowledge of the bound conformation of this novel inhibitor provides a template and future direction for the design of new classes of FPTase antagonists.     

Specific isoprenoid modification is required for function of normal, but not oncogenic, Ras protein.

While the Ras C-terminal CAAX sequence signals modification by a 15-carbon farnesyl isoprenoid, the majority of isoprenylated proteins in mammalian cells are modified instead by a 20-carbon geranylgeranyl moiety. To determine the structural and functional basis for modification of proteins by a specific isoprenoid group, we have generated chimeric Ras proteins containing C-terminal CAAX sequences (CVLL and CAIL) from geranylgeranyl-modified proteins and a chimeric Krev-1 protein containing the H-Ras C-terminal CAAX sequence (CVLS). Our results demonstrate that both oncogenic Ras transforming activity and Krev-1 antagonism of Ras transforming activity can be promoted by either farnesyl or geranylgeranyl modification. Similarly, geranylgeranyl-modified normal Ras [Ras(WT)CVLL], when overexpressed, exhibited the same level of transforming activity as the authentic farnesyl-modified normal Ras protein. Therefore, farnesyl and geranylgeranyl moieties are functionally interchangeable for these biological activities. In contrast, expression of moderate levels of geranylgeranyl-modified normal Ras inhibited the growth of untransformed NIH 3T3 cells. This growth inhibition was overcome by coexpression of the mutant protein with oncogenic Ras or Raf, but not with oncogenic Src or normal Ras. The similar growth-inhibiting activities of Ras(WT)CVLL and the previously described Ras(17N) dominant inhibitory mutant suggest that geranylgeranyl-modified normal Ras may exert its growth-inhibiting action by perturbing endogenous Ras function. These results suggest that normal Ras function may specifically require protein modification by a farnesyl, but not a geranylgeranyl, isoprenoid.     

LC-NMR: a new tool to expedite the dereplication and identification of natural products

The rapid identification of known or undesirable compounds from natural products extracts — “dereplication” — is an important step in an efficiently run natural products discovery program. Dereplication strategies use analytical techniques and database searching to determine the identity of an active compound at the earliest possible stage in the discovery process. In the past few years, advances in technology have allowed the development of tandem analytical techniques such as liquid chromatography mass spectrometry (LC-MS), LC-MS-MS, liquid chromatography nuclear magnetic resonance (LC-NMR), and LC-NMR-MS. LC-NMR, despite its lower sensitivity as compared to LC-MS, provides a powerful tool for rapid identification of known compounds and identification of structure classes of novel compounds. LC-NMR is especially useful in instances where the data from LC-MS are incomplete or do not allow confident identification of the active component of a sample. LC-NMR has been used to identify the marine alkaloid aaptamine as the active component in an extract of the sponge Aaptos sp. This extract had been identified as an enzyme inhibitor by a high throughput screening (HTS) effort. Isolated aaptamine exhibited an IC₅₀=120 μM against this enzyme. Strategies for the identification of aaptamine and for the use of LC-NMR in a natural products HTS program are discussed. Journal of Industrial Microbiology & Biotechnology (2000) 25, 342–345. Received 30 March 2000/ Accepted in revised form 03 July 2000     

Involvement of farnesyl protein transferase (FPTase) in FcarepsilonRI-induced activation of RBL-2H3 mast cells

Changes in farnesyl protein transferase (FPTase) activity and FPTase beta-subunit protein levels were determined in IgE-sensitized RBL-2H3 mast cells in response to polyvalent antigen administration. Ten minutes after the addition of DNP modified BSA to mast cells, whose high affinity receptor for IgE (FcvarepsilonRI) contained bound anti-DNP IgE, FPTase specific activity increased by 54 +/- 28%. Time course studies showed FPTase specific activity doubled during a 20- to 30-min period after antigen-induced cell aggregation. Also, an increase in FPTase beta-subunit protein during this time ( approximately 30%) was observed; this protein increase was not accompanied by a similar increase in FPTase beta-subunit m-RNA levels. The FcvarepsilonRI aggregation had no significant effect on the activities of other enzymes involved with farnesyl diphosphate (FPP) metabolism: FPP synthase, isopentenyl diphosphate isomerase, geranylgeranyl protein transferase, and squalene synthase. Specific inhibition of FPTase activity by manumycin was studied to determine what role FPTase plays in mast cell activation. Manumycin profoundly inhibited hexosaminidase release in activated cells, indicating FPTase is required for signal transduction involved with protein exocytosis from RBL-2H3 mast cells.     

High-performance liquid chromatography/mass spectrometry characterization of Ki4B-Ras in PSN-1 cells treated with the prenyltransferase inhibitor L-778,123

Cellular transformation by Ras oncoproteins requires the posttranslation modification of farnesylation in a reaction catalyzed by farnesyl protein transferase (FPTase). Thus, inhibitors of FPTase have been developed as potential anticancer agents. However, recent studies with selective inhibitors of FPTase have shown that Ki4B-Ras retains its ability to transform cells by undergoing alternative prenylation by the related geranylgeranyl protein transferase I (GGPTase-I) in human tumor cells. We have developed a high-performance liquid chromatography/mass spectrometry assay for the detection and quantitation of the different processing states of Ki4B-Ras isolated from PSN-1 cells (a human pancreatic cell line with an activating Gly12 to Arg mutation) treated with the prenyltransferase inhibitor, L-778,123. Recently tested in the clinic, L-778,123 is a potent inhibitor of FPTase (in vitro IC50 = 2 nM) with some activity against GGPTase-I (in vitro IC50 = 98 nM). We find primarily farnesylated-Ki4B-Ras in vehicle-treated PSN-1 cells, a mixture of farnesylated- and geranylgeranylated-Ki4B-Ras in cells treated with nanomolar concentrations of L-778,123, and a mixture of unprocessed, farnesylated, and geranylgeranylated-Ki4B-Ras in cells treated with micromolar concentrations of compound. Of importance, this technique does not require metabolic labeling and may be used as a pharmacodynamic assay for Ki4B-Ras processing in mouse models.     

海南海口温泉真菌、细菌多样性及其环境影响因素分析

【目的】海南海口含有丰富的温泉资源,对温泉微生物多样性进行研究,有助于进一步开发和利用海南温泉微生物资源。【方法】本文采用Illumina Hi Seq高通量测序技术对海口3个温泉[海甸岛荣域温泉(S1)、火山口开心农场温泉(S2)和西海岸海长流温泉(S3)]水样中微生物ITS序列和16Sr RNA基因V3-V4区进行测序及生物信息学分析,探究海口市3个不同区域的温泉真菌多样性与细菌多样性。【结果】(1)α多样性分析表明,真菌群落中,S3(29)S1(29)S2,而在细菌群落中,S2(29)S1(29)S3。β多样性分析表明,3个温泉真菌群落和细菌群落组成差异皆显著。(2)分类分析表明,温泉真菌群落优势菌门为子囊菌门(Ascomycota)和担子菌门(Basidiomycota),细菌群落优势菌门为变形菌门(Proteobacteria)、拟杆菌门(Bacteroidetes)、Thermi、硝化螺旋菌门(Nitrospirae)、绿菌门(Chlorobi)、厚壁菌门(Firmicutes)、绿弯菌门(Chloroflexi)、放线菌门(Actinobacteria)。(3) CCA (Canonical correspondence analysis)分析表明,3个温泉的真菌群落主要影响因子是温度,细菌群落主要影响因子是总磷。【结论】海南省海口市温泉中含有丰富的微生物资源,其微生物群落组成受多种环境因子影响,且影响真菌和细菌的主要环境因子不同。     

Composition and in vitro antimicrobial activity of Populus buds and poplar-type propolis

The antibacterial activity of propolis has been widely investigated. Since reports dealing with antimicrobial activity of the origin of propolis are not available, this study was carried out aiming to analyse the in vitro antimicrobial activity of the methanol extracts of poplar type propolis and Populus (Populus nigra, P. alba, P. tremuloides) buds as its sources against standard strains of a panel of microorganisms by determining the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). The concentrations of the “poplar” phenolics were relatively high (4.5%) and some compounds typical for P. nigra such as pinobanksin and 4,3 acetyloxycaffeate were found in the propolis sample by GC-MS. The poplar type propolis and Populus bud exudates were found to inhibit most clinically important microorganisms in a wide spectrum including pathogenic yeasts but not Gram-negative bacteria.