Protein disulfide isomerase assisted protein folding in malaria parasites

In eukaryotes, the formation of protein disulfide bonds among cysteine residues is mediated by protein disulfide isomerases and occurs in the highly oxidised environment of the endoplasmic reticulum. This process is poorly understood in malaria parasites. In this paper, we report the gene isolation, sequence and phylogenetic comparisons, protein structure and thioredoxin-domain analyses of nine protein disulfide isomerases-like molecules from five species of malaria parasites including Plasmodium falciparum and Plasmodium vivax (human), Plasmodium knowlesi (simian) and Plasmodium berghei and Plasmodium yoelii (murine). Four of the studied protein disulfide isomerases belong to P. falciparum malaria and have been named PfPDI-8, PfPDI-9, PfPDI-11 and PfPDI-14, based on their chromosomal location. Among these, PfPDI-8 bears the closest similarity to a prototype PDI molecule with two thioredoxin domains (containing CGHC active sites) and a C-terminal Endoplasmic reticulum retrieval signal, SEEL. PfPDI-8 is expressed during all stages of parasite life cycle and is highly conserved (82-96% identity at amino acid level) in the other four Plasmodium species studied. Detailed biochemical analysis of PfPDI-8 revealed that this molecule is a potent oxido-reductase enzyme that facilitated the disulfide-dependent conformational folding of EBA-175, a leading malaria vaccine candidate. These studies open the avenues to understand the process of protein folding and secretory pathway in malaria parasites that in turn might aid in the production of superior recombinant vaccines and provide novel drug targets.     

Hypoxia and hypoxia mimetic cooperate to counteract tumor cell resistance to glucose starvation preferentially in tumor cells with mutant p53

We demonstrated that exogenous pyruvate promotes survival under glucose depletion in aerobic mutant p53 (R175H) human melanoma cells. Others subsequently indicated that mutant p53 tumor cells undergo p53 degradation and cell death under aerobic glucose-free conditions. Since glucose starvation occurs in hypoxic gradients of poorly vascularized tumors, we investigated the role of p53 siRNA under hypoxia in wt p53 C8161 melanoma using glucose starvation or 5 mM physiological glucose. p53 Silencing decreased survival of glucose-starved C8161 melanoma with pyruvate supplementation under hypoxia (?1% oxygen), but increased resistance to glycolytic inhibitors oxamate and 2-deoxyglucose in 5 mM glucose, preferentially under normoxia. Aiming to counteract hypoxic tumor cell survival irrespective of p53 status, genetically-matched human C8161 melanoma harboring wt p53 or mutant p53 (R175H) were used combining true hypoxia (?1% oxygen) and hypoxia mimetic CoCl₂. No significant decrease in metabolic activity was evidenced in C8161 melanoma irrespective of p53 status in 2.5 mM glucose after 48 h of physical hypoxia. However, combining the latter with 100 μM CoCl₂ was preferentially toxic for mutant p53 C8161 melanoma, and was enhanced by catalase in wt p53 C8161 cells. Downregulation of MnSOD and LDHA accompanied the toxicity induced by hypoxia and CoCl₂ in 5 mM glucose, and these changes were enhanced by oxamate or 2-deoxyglucose. Our results show for the first time that survival of malignant cells in a hypoxic microenvironment can be counteracted by hypoxia mimetic co-treatment in a p53 dependent manner.     

Flt3 mutation activates p21WAF1/CIP1 gene expression through the action of STAT5

Metastin, a post-translationally modified variant of KiSS1, was recently identified as an endogenous peptide agonist for a novel G-protein coupled receptor, hOT7T175 (AXOR12, GPR54). In this study, we analyzed the role of KiSS1 and hOT7T175 in both pancreatic cancer tissues and pancreatic cancer cell lines. Furthermore, we synthesized novel short variant forms of metastin and tested the inhibitory effect of those variants on in vitro cell functions that are relevant to metastasis. Pancreatic cancer tissues showed significantly lower expression of KiSS1 mRNA than normal tissues (p=0.018), while cancer tissues showed significantly higher expression of hOT7T175 mRNA than normal pancreatic tissues (p=0.027). In human pancreatic cancer cell lines, KiSS1 mRNA was highly expressed in 2 out of 6 pancreatic cancer cell lines, while hOT7T175 mRNA was expressed in all cell lines at various degrees. PANC-1 cells showed the highest expression of hOT7T175. Exogenous metastin did not suppress cell proliferation but significantly reduced the in vitro migration of PANC-1 cells (p<0.01). Metastin induced activation of ERK1 in PANC-1 and AsPC-1 cells. Finally, we synthesized 3 novel short variant forms of metastin, FM053a2TFA, FM059a2TFA, and FM052a4TFA. These metastin variants significantly suppressed the migration of PANC-1 cells and activated ERK1. These data suggest that the metastin receptor, hOT7T175, is one of the promising targets for suppression of metastasis, and that small metastin variants could be an anti-metastatic agent to pancreatic cancer.     

Reconstitution of beta-carotene hydroxylase activity of thermostable CYP175A1 monooxygenase

CYP175A1 is a thermostable P450 Monooxygenase from Thermus thermophilus HB27, demonstrating in vivo activity towards beta-carotene. Activity of CYP175A1 was reconstituted in vitro using artificial electron transport proteins. First results were obtained in the mixture with a crude Escherichia coli cell extract at 37 degrees C. In this system, beta-carotene was hydroxylated to beta-cryptoxanthin. The result indicated the presence of electron transport enzymes among the E. coli proteins, which are suitable for CYP175A1. However, upon in vitro reconstitution of CYP175A1 activity with purified recombinant flavodoxin and flavodoxin reductase from E. coli, only very low beta-cryptoxanthin production was observed. Remarkably, with another artificial electron transport system, putidaredoxin and putidaredoxin reductase from Pseudomonas putida, purified CYP175A1 enzyme hydroxylated beta-carotene at 3- and also 3'-positions, resulting in beta-cryptoxanthin and zeaxanthin. Under the optimal reaction conditions, the turnover rate of the enzyme reached 0.23 nmol beta-cryptoxanthin produced per nmol P450 per min.     

The solution structure of the adhesion protein Bd37 from Babesia divergens reveals structural homology with eukaryotic proteins involved in membrane trafficking

Babesia divergens is the Apicomplexa agent of the bovine babesiosis in Europe: this infection leads to growth and lactation decrease, so that economical losses due to this parasite are sufficient to require the development of a vaccine. The major surface antigen of B. divergens has been described as a 37 kDa protein glycosyl phosphatidyl inositol (GPI)-anchored at the surface of the merozoite. The immuno-prophylactic potential of Bd37 has been demonstrated, and we present here the high-resolution solution structure of the 27 kDa structured core of Bd37 (Δ-Bd37) using NMR spectroscopy. A model for the whole protein has been obtained using additional small angle X-ray scattering (SAXS) data. The knowledge of the 3D structure of Bd37 allowed the precise epitope mapping of antibodies on its surface. Interestingly, the geometry of Δ-Bd37 reveals an intriguing similarity with the exocyst subunit Exo84p C-terminal region, an eukaryotic protein that has a direct implication in vesicle trafficking. This strongly suggests that Apicomplexa have developed in parallel molecular machines similar in structure and function to the ones used for endo- and exocytosis in eukaryotic cells.     

Specific killing of DNA damage-response deficient cells with inhibitors of poly(ADP-ribose) glycohydrolase

Poly(ADP-ribosylation) of proteins following DNA damage is well studied and the use of poly(ADP-ribose) polymerase (PARP) inhibitors as therapeutic agents is an exciting prospect for the treatment of many cancers. Poly(ADP-ribose) glycohydrolase (PARG) has endo- and exoglycosidase activities which can cleave glycosidic bonds, rapidly reversing the action of PARP enzymes. Like addition of poly(ADP-ribose) (PAR) by PARP, removal of PAR by PARG is also thought to be required for repair of DNA strand breaks and for continued replication at perturbed forks. Here we use siRNA to show a synthetic lethal relationship between PARG and BRCA1, BRCA2, PALB2, FAM175A (ABRAXAS) and BARD1. In addition, we demonstrate that MCF7 cells depleted of these proteins are sensitive to Gallotannin and a novel and specific PARG inhibitor PDD00017273. We confirm that PARG inhibition increases endogenous DNA damage, stalls replication forks and increases homologous recombination, and propose that it is the lack of homologous recombination (HR) proteins at PARG inhibitor-induced stalled replication forks that induces cell death. Interestingly not all genes that are synthetically lethal with PARP result in sensitivity to PARG inhibitors, suggesting that although there is overlap, the functions of PARP and PARG may not be completely identical. These data together add further evidence to the possibility that single treatment therapy with PARG inhibitors could be used for treatment of certain HR deficient tumours and provide insight into the relationship between PARP, PARG and the processes of DNA repair.     

Dopamine-β-hydroxylase-, neurotensin-, substance P-, vasoactive intestinal polypeptide- and enkephalin-immunohistochemistry of paravertebral and prevertebral ganglia in the cat

Summary Para and prevertebral ganglia of the cat were investigated for immunoreactivity (IR) against neurotensin (NT), vasoactive intestinal polypeptide (VIP), substance P (SP) and enkephalin (ENK). Dopamine--hydroxylase- (DBH)-IR was studied in consecutive sections to correlate the distribution of noradrenergic/adrenergic neurons with that of peptidergic nerve fibres and cells.In paravertebral (cervical and thoracic) ganglia, NT-IR or ENK-IR nerve fibres were seen in areas in which DBH-IR fibre networks also occurred. NT-IR varicosities were often in close contact with perikarya of principal ganglionic cells on which DBH-IR varicosities also terminated. Such an association was rarely seen between ENK-IR and DBH-IR fibre baskets. NT-IR and ENK-IR fibre baskets were not found to occur around the same principal ganglionic cell. The distribution of VIP-IR and SP-IR nerve fibres did not coincide with that of DBH-IR fibres.In prevertebral ganglia (celiac-superior mesenteric and inferior mesenteric) DBH-IR or VIP-IR varicosities surrounded the majority of principal ganglionic neurons. ENK-IR or SP-IR fibres were closely associated with only a minority of the neurons; NT-IR networks were rather sparse. Some principal neurons were approached by DBH-IR fibres and by different peptide-IR fibres.In paravertebral ganglia some principal ganglionic cells contained VIP-IR, a few of which were also surrounded by NT-IR varicosities. VIP-IR perikarya in prevertebral ganglia were extremely rare. No NT-IR, SP-IR or ENK-IR principal ganglionic cells were found.Glomus-like paraganglionic cell clusters in paravertebral and prevertebral ganglia exhibited DBH-IR cell bodies. Moreover, the clusters also contained ENK-IR or SP-IR cells. NT-IR varicosities were observed adjacent to clustered paraganglionic cells. Only few singly located paraganglionic cells were NT-IR or ENK-IR.The differential distribution of peptide-IR nerve endings in the investigated ganglia suggests a regulation of impulse transmission that seems to be related to the target organs.Fellow of the Heisenberg foundationSupported by the DFG, grants He 919/5, Re 520/1-2, and SFB 90 Carvas, Heidelberg     

Glutathione transferase Omega 1-1 (GSTO1-1) modulates Akt and MEK1/2 signaling in human neuroblastoma cell SH-SY5Y

In the human neuroblastoma SH-SY5Y cell line, the glutathione transferase Omega 1-1 (GSTO1-1) appears to modulate Akt and MEK1/2 kinase activation. We observed a glutathionylation modification was involved in the activation of Akt but not MEK1/2. With the specific GSTO1-1 inhibitor ML175, we show the enzyme activity of GSTO1-1 is important for modulation as the inhibited GSTO1-1 allowed activation of both Akt and MEK1/2. The inhibition of GSTO1-1 showed a similar extent of activation of Akt and MEK1/2 as treatment by the endotoxin lipopolysaccharide. The GSTO1-1 also either directly interacts with Akt and MEK1/2 or interacts with a protein complexed with Akt and MEK1/2 as both kinases coimmunoprecipitated with GSTO1-1. The results suggest that GSTO1-1 enzyme activity inhibits the activation of these two kinases to maintain basal levels. The possible regulation by GSTO1-1 is of interest as both kinases have hundreds of potential downstream targets that are known to have contributions to various cellular processes including survival, growth, proliferation, and metabolism.