The Protein quality control system manages plant defence compound synthesis

Jasmonates are ubiquitous oxylipin-derived phytohormones that are essential in the regulation of many development, growth and defence processes. Across the plant kingdom, jasmonates act as elicitors of the production of bioactive secondarymetabolites that serve in defence against attackers. Knowledge of the conserved jasmonate perception and early signalling machineries is increasing, but the downstream mechanisms that regulate defence metabolism remain largely unknown. Herewe showthat, in the legumeMedicago truncatula, jasmonate recruits the endoplasmic-reticulum-associated degradation (ERAD)quality control system tomanagethe production of triterpene saponins, widespread bioactive compounds that share a biogenic origin with sterols. An ERAD-type RING membraneanchor E3 ubiquitin ligase is co-expressed with saponin synthesis enzymes to control the activity of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), the rate-limiting enzyme in the supply of the ubiquitous terpene precursor isopentenyl diphosphate. Thus, unrestrained bioactive saponin accumulationis prevented and plant development and integrity secured. This control apparatus is equivalent to the ERAD system that regulates sterol synthesis in yeasts and mammals but that uses distinct E3 ubiquitin ligases, of the HMGR degradation 1 (HRD1) type, to direct destruction of HMGR. Hence, the general principles for the management of sterol and triterpene saponin biosynthesis are conserved across eukaryotes but can be controlled by divergent regulatory cues.

The Protein quality control system manages plant defence compound synthesis

Jasmonates are ubiquitous oxylipin-derived phytohormones that are essential in the regulation of many development, growth and defence processes. Across the plant kingdom, jasmonates act as elicitors of the production of bioactive secondarymetabolites that serve in defence against attackers. Knowledge of the conserved jasmonate perception and early signalling machineries is increasing, but the downstream mechanisms that regulate defence metabolism remain largely unknown. Herewe showthat, in the legumeMedicago truncatula, jasmonate recruits the endoplasmic-reticulum-associated degradation (ERAD)quality control system tomanagethe production of triterpene saponins, widespread bioactive compounds that share a biogenic origin with sterols. An ERAD-type RING membraneanchor E3 ubiquitin ligase is co-expressed with saponin synthesis enzymes to control the activity of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), the rate-limiting enzyme in the supply of the ubiquitous terpene precursor isopentenyl diphosphate. Thus, unrestrained bioactive saponin accumulationis prevented and plant development and integrity secured. This control apparatus is equivalent to the ERAD system that regulates sterol synthesis in yeasts and mammals but that uses distinct E3 ubiquitin ligases, of the HMGR degradation 1 (HRD1) type, to direct destruction of HMGR. Hence, the general principles for the management of sterol and triterpene saponin biosynthesis are conserved across eukaryotes but can be controlled by divergent regulatory cues.

Combinatorial Synthesis and Biological Evaluation of Inhibitors of D-Ala-D-Ala-Ligase

Report for the scientific sojourn carried out at the Max Planck Institut of Molecular Phisiology, Germany, from 2006 to 2008.The work carried out during this postdoctoral stage was focused on two different projects. Firstly, identification of D-Ala D-Ala Inhibitors and the development of new synthethic approaches to obtain lipidated peptides and proteins and the use of these lipidated proteins in biological and biophysical studies. In the first project, new D-Ala D-Ala inhibitors were identified by using structural alignments of the ATP binding sites of the bacterial ligase DDl and protein and lipid kinases in complex with ATP analogs. We tested a series of commercially available kinase inhibitors and found LFM-A13 and Tyrphostine derivatives to inhibit DDl enzyme activity. Based on the initial screening results we synthesized a series of malononitrilamide and salicylamide derivatives and were able to confirm the validity of these scaffolds as inhibitors of DDl. From this investigation we gained a better understanding of the structural requirements and limitations necessary for the preparation of ATP competitive DDl inhibitors. The compounds in this study may serve as starting points for the development of bi-substrate inhibitors that incorporate both, an ATP competitive and a substrate competitive moiety. Bisubstrate inhibitors that block the ATP and D-Ala binding sites should exhibit enhanced selectivity and potency profiles by preferentially inhibiting DDl over kinases. In the second project, an optimized synthesis for tha alkylation of cysteins using the thiol ene reaction was establisehd. This new protocol allowed us to obtain large amounts of hexadecylated cysteine that was required for the synthesis of differently lipidated peptides. Afterwards the synthesis of various N-ras peptides bearing different lipid anchors was performed and the peptides were ligated to a truncated N-ras protein. The influence of this differently lipidated N-ras proteins on the partioning and association of N-Ras in model membrane subdomains was studied using Atomic Force Microscopy.

A short motif in Drosophila SECIS Binding Protein 2 provides differential binding affinity to SECIS RNA hairpins

Selenoproteins contain the amino acid selenocysteine which is encoded by a UGA Sec codon. Recoding UGA Sec requires a complex mechanism, comprising the cis-acting SECIS RNA hairpin in the 3′UTR of selenoprotein mRNAs, and trans-acting factors. Among these, the SECIS Binding Protein 2 (SBP2) is central to the mechanism. SBP2 has been so far functionally characterized only in rats and humans. In this work, we report the characterization of the Drosophila melanogaster SBP2 (dSBP2). Despite its shorter length, it retained the same selenoprotein synthesis-promoting capabilities as the mammalian counterpart. However, a major difference resides in the SECIS recognition pattern: while human SBP2 (hSBP2) binds the distinct form 1 and 2 SECIS RNAs with similar affinities, dSBP2 exhibits high affinity toward form 2 only. In addition, we report the identification of a K (lysine)-rich domain in all SBP2s, essential for SECIS and 60S ribosomal subunit binding, differing from the well-characterized L7Ae RNA-binding domain. Swapping only five amino acids between dSBP2 and hSBP2 in the K-rich domain conferred reversed SECIS-binding properties to the proteins, thus unveiling an important sequence for form 1 binding.

Tumor necrosis factor-alpha mediates changes in tissue protein turnover in a rat cancer cachexia model.

Rats bearing the Yoshida AH-130 ascites hepatoma showed enhanced fractional rates of protein degradation in gastrocnemius muscle, heart, and liver, while fractional synthesis rates were similar to those in non-tumor bearing rats. This hypercatabolic pattern was associated with marked perturbations of the hormonal homeostasis and presence of tumor necrosis factor in the circulation. The daily administration of a goat anti-murine TNF IgG to tumor-bearing rats decreased protein degradation rates in skeletal muscle, heart, and liver as compared with tumor-bearing rats receiving a nonimmune goat IgG. The anti-TNF treatment was also effective in attenuating early perturbations in insulin and corticosterone homeostasis. Although these results suggest that tumor necrosis factor plays a significant role in mediating the changes in protein turnover and hormone levels elicited by tumor growth, the inability of such treatment to prevent a reduction in body weight implies that other mediators or tumor-related events were also involved.

A T42A Ran Mutation: Differential Interactions with Effectors and Regulators, and Defect in Nuclear Protein Import

Ran, the small, predominantly nuclear GTPase, has been implicated in the regulation of a variety of cellular processes including cell cycle progression, nuclear-cytoplasmic trafficking of RNA and protein, nuclear structure, and DNA synthesis. It is not known whether Ran functions directly in each process or whether many of its roles may be secondary to a direct role in only one, for example, nuclear protein import. To identify biochemical links between Ran and its functional target(s), we have generated and examined the properties of a putative Ran effector mutation, T42A-Ran. T42A-Ran binds guanine nucleotides as well as wild-type Ran and responds as well as wild-type Ran to GTP or GDP exchange stimulated by the Ran-specific guanine nucleotide exchange factor, RCC1. T42A-Ran·GDP also retains the ability to bind p10/NTF2, a component of the nuclear import pathway. In contrast to wild-type Ran, T42A-Ran·GTP binds very weakly or not detectably to three proposed Ran effectors, Ran-binding protein 1 (RanBP1), Ran-binding protein 2 (RanBP2, a nucleoporin), and karyopherin ß (a component of the nuclear protein import pathway), and is not stimulated to hydrolyze bound GTP by Ran GTPase-activating protein, RanGAP1. Also in contrast to wild-type Ran, T42A-Ran does not stimulate nuclear protein import in a digitonin permeabilized cell assay and also inhibits wild-type Ran function in this system. However, the T42A mutation does not block the docking of karyophilic substrates at the nuclear pore. These properties of T42A-Ran are consistent with its classification as an effector mutant and define the exposed region of Ran containing the mutation as a probable effector loop.

Anti-inflammatory effects of pancreatitis associated protein in inflammatory bowel disease

Background and aims: Increased pancreatitis associated protein (PAP) mRNA has been reported in active inflammatory bowel disease (IBD). The aims of the current study were to characterise PAP production in IBD and the effects of PAP on inflammation. Patients and methods: Serum PAP levels were determined in healthy controls (n¿=¿29), inflammatory controls (n¿=¿14), and IBD patients (n¿=¿171). Ex vivo PAP secretion in intestinal tissue was measured in 56 IBD patients and 13 healthy controls. Cellular origin of PAP was determined by immunohistochemistry. The effects of exogenous PAP on nuclear factor ¿B (NF¿B) activation, proinflammatory cytokine production, and endothelial adhesion molecule expression were also analysed ex vivo. Results: Patients with active IBD had increased serum PAP levels compared with controls, and these levels correlated with clinical and endoscopic disease severity. Ex vivo intestinal PAP synthesis was increased in active IBD and correlated with endoscopic and histological severity of inflammatory lesions. PAP localised to colonic Paneth cells. Incubation of mucosa from active Crohn¿s disease with PAP dose dependently reduced proinflammatory cytokines secretion. PAP prevented TNF-¿ induced NF¿B activation in monocytic, epithelial, and endothelial cells and reduced proinflammatory cytokine mRNA levels and adhesion molecule expression. Conclusions: PAP is synthesised by Paneth cells and is overexpressed in colonic tissue of active IBD. PAP inhibits NF¿B activation and downregulates cytokine production and adhesion molecule expression in inflamed tissue. It may represent an anti-inflammatory mechanism and new therapeutic strategy in IBD.

Tumor necrosis factor-alpha mediates changes in tissue protein turnover in a rat cancer cachexia model.

Rats bearing the Yoshida AH-130 ascites hepatoma showed enhanced fractional rates of protein degradation in gastrocnemius muscle, heart, and liver, while fractional synthesis rates were similar to those in non-tumor bearing rats. This hypercatabolic pattern was associated with marked perturbations of the hormonal homeostasis and presence of tumor necrosis factor in the circulation. The daily administration of a goat anti-murine TNF IgG to tumor-bearing rats decreased protein degradation rates in skeletal muscle, heart, and liver as compared with tumor-bearing rats receiving a nonimmune goat IgG. The anti-TNF treatment was also effective in attenuating early perturbations in insulin and corticosterone homeostasis. Although these results suggest that tumor necrosis factor plays a significant role in mediating the changes in protein turnover and hormone levels elicited by tumor growth, the inability of such treatment to prevent a reduction in body weight implies that other mediators or tumor-related events were also involved.

Synthesis and multi-target biological profiling of a novel family of rhein derivatives as disease-modifying anti-Alzheimer agents

We have synthesized a family of rheinhuprine hybrids to hit several key targets for Alzheimer"s disease. Biological screening performed in vitro and in Escherichia coli cells has shown that these hybrids exhibit potent inhibitory activities against human acetylcholinesterase butyrylcholinesterase, and BACE-1, dual Aβ42 and tau anti-aggregating activity, and brain permeability. Ex vivo studies with the leads (+)- and ()-7e in brain slices of C57bl6 mice have revealed that they efficiently protect against the Aβ-induced synaptic dysfunction , preventing the loss of synaptic proteins and/or have a positive effect on the induction of long term potentiation. In vivo studies in APP-PS1 transgenic mice treated i.p. for 4 weeks with (+)- and ()-7e have shown a central soluble Aβ lowering effect, accompanied by an increase in the levels of mature amyloid precursor protein (APP). Thus, (+)- and ()-7e emerge as very promising disease-modifying anti-Alzheimer drug candidates.

Synthesis and multi-target biological profiling of a novel family of rhein derivatives as disease-modifying anti-Alzheimer agents

We have synthesized a family of rheinhuprine hybrids to hit several key targets for Alzheimer"s disease. Biological screening performed in vitro and in Escherichia coli cells has shown that these hybrids exhibit potent inhibitory activities against human acetylcholinesterase butyrylcholinesterase, and BACE-1, dual Aβ42 and tau anti-aggregating activity, and brain permeability. Ex vivo studies with the leads (+)- and ()-7e in brain slices of C57bl6 mice have revealed that they efficiently protect against the Aβ-induced synaptic dysfunction , preventing the loss of synaptic proteins and/or have a positive effect on the induction of long term potentiation. In vivo studies in APP-PS1 transgenic mice treated i.p. for 4 weeks with (+)- and ()-7e have shown a central soluble Aβ lowering effect, accompanied by an increase in the levels of mature amyloid precursor protein (APP). Thus, (+)- and ()-7e emerge as very promising disease-modifying anti-Alzheimer drug candidates.

Insertion of a malE B-Galactosidase fusion protein into the envelope of Escherichia coli disrupts biogenesis of outer membrane proteins and processing of inner membrane proteins

The synthesis of a membrane-bound MalE ,B-galactosidase hybrid protein, when induced by growth of Escherichia coli on maltose, leads to inhibition of cell division and eventually a reduced rate of mass increase. In addition, the relative rate of synthesis of outer membrane proteins, but not that of inner membrane proteins, was reduced by about 50%o. Kinetic experiments demonstrated that this reduction coincided with the period of maximum synthesis of the hybrid protein (and another maltose-inducible protein, LamB). The accumulation of this abnormal protein in the envelope therefore appeared specifically to inhibit the synthesis, the assembly of outer membrane proteins, or both, indicating that the hybrid protein blocks some export site or causes the sequestration of some limiting factor(s) involved in the export process. Since the MalE protein is normally located in the periplasm, the results also suggest that the synthesis of periplasmic and outer membrane proteins may involve some steps in common. The reduced rate of synthesis of outer membrane proteins was also accompanied by the accumulation in the envelope of at least one outer membrane protein and at least two inner membrane proteins as higher-molecular-weight forms, indicating that processing (removal of the N-terminal signal sequence) was also disrupted by the presence of the hybrid protein. These results may indicate that the assembly of these membrane proteins is blocked at a relatively late step rather than at the level of primary recognition of some site by the signal sequence. In addition, the results suggest that some step common to the biogenesis of quite different kinds of envelope protein is blocked by the presence of the hybrid protein.

Sterol regulatory element binding protein-1a transactivates 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase gene promoter

6-Phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB)catalyzes the synthesis and degradation of fructose-2,6-bisphosphate, a key modulator of glycolysis-gluconeogenesis. To gain insight into the molecular mechanism behind hormonal and nutritional regulation of PFKFB expression, we have cloned and characterized the proximal promoter region of the liver isoform of PFKFB (PFKFB1) from gilthead sea bream (Sparus aurata). Transient transfection of HepG2 cells with deleted gene promoter constructs and electrophoretic mobility shift assays allowed us to identify a sterol regulatory element (SRE) to which SRE binding protein-1a (SREBP-1a)binds and transactivates PFKFB1 gene transcription. Mutating the SRE box abolished SREBP-1a binding and transactivation. The in vivo binding of SREBP-1a to the SRE box in the S. aurata PFKFB1 promoter was confirmed by chromatin immunoprecipitation assays. There is a great deal of evidence for a postprandial rise of PFKB1 mRNA levels in fish and rats. Consistently, starved-to-fed transition and treatment with glucose or insulin increased SREBP-1 immunodetectable levels, SREBP-1 association to PFKFB1 promoter, and PFKFB1 mRNA levels in the piscine liver. Our findings demonstrate involvement of SREBP-1a in the transcriptional activation of PFKFB1, and we conclude that SREBP-1a may exert a key role mediating postprandial activation of PFKFB1 transcription.

Sterol regulatory element binding protein-1a transactivates 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase gene promoter

6-Phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB)catalyzes the synthesis and degradation of fructose-2,6-bisphosphate, a key modulator of glycolysis-gluconeogenesis. To gain insight into the molecular mechanism behind hormonal and nutritional regulation of PFKFB expression, we have cloned and characterized the proximal promoter region of the liver isoform of PFKFB (PFKFB1) from gilthead sea bream (Sparus aurata). Transient transfection of HepG2 cells with deleted gene promoter constructs and electrophoretic mobility shift assays allowed us to identify a sterol regulatory element (SRE) to which SRE binding protein-1a (SREBP-1a)binds and transactivates PFKFB1 gene transcription. Mutating the SRE box abolished SREBP-1a binding and transactivation. The in vivo binding of SREBP-1a to the SRE box in the S. aurata PFKFB1 promoter was confirmed by chromatin immunoprecipitation assays. There is a great deal of evidence for a postprandial rise of PFKB1 mRNA levels in fish and rats. Consistently, starved-to-fed transition and treatment with glucose or insulin increased SREBP-1 immunodetectable levels, SREBP-1 association to PFKFB1 promoter, and PFKFB1 mRNA levels in the piscine liver. Our findings demonstrate involvement of SREBP-1a in the transcriptional activation of PFKFB1, and we conclude that SREBP-1a may exert a key role mediating postprandial activation of PFKFB1 transcription.

Functional and evolutionary analysis of DXL1, a non-essential gene encoding a 1-deoxy-D-xylulose 5-phosphate synthase like protein in Arabidopsis thaliana

The synthesis of 1-deoxy-D-xylulose 5-phosphate (DXP), catalyzed by the enzyme DXP synthase (DXS), represents a key regulatory step of the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway for isoprenoid biosynthesis. In plants DXS is encoded by small multigene families that can be classified into, at least, three specialized subfamilies. Arabidopsis thaliana contains three genes encoding proteins with similarity to DXS, including the well-known DXS1/CLA1 gene, which clusters within subfamily I. The remaining proteins, initially named DXS2 and DXS3, have not yet been characterized. Here we report the expression and functional analysis of A. thaliana DXS2. Unexpectedly, the expression of DXS2 failed to rescue Escherichia coli and A. thaliana mutants defective in DXS activity. Coherently, we found that DXS activity was negligible in vitro, being renamed as DXL1 following recent nomenclature recommendation. DXL1 is targeted to plastids as DXS1, but shows a distinct expression pattern. The phenotypic analysis of a DXL1 defective mutant revealed that the function of the encoded protein is not essential for growth and development. Evolutionary analyses indicated that DXL1 emerged from DXS1 through a recent duplication apparently specific of the Brassicaceae lineage. Divergent selective constraints would have affected a significant fraction of sites after diversification of the paralogues. Furthermore, amino acids subjected to divergent selection and likely critical for functional divergence through the acquisition of a novel, although not yet known, biochemical function, were identified. Our results provide with the first evidences of functional specialization at both the regulatory and biochemical level within the plant DXS family.

Synthesis of a tetrahydroimidazo-[2',1':2,3]thiazolo[5,4-c]pyridine derivative with Met inhibitory activity

A straightforward synthesis of the Met antagonist JLK1360 involving an alkylationcyclocondensation process using aminothiazole 1 and nitrophenacyl bromide 2, reduction of the nitro group, and coupling of the resulting tetracyclic aniline 5 with an appropriate N-acyl alanine derivative, is reported.