Band 390, Heft 12

Tumor growth is dependent on several key factors. Apart from immune escape and an efficient blockade of apoptotic signals, tumors require oxygen and nutrients to grow past a diameter of 2 μm. Therefore, it is of vital importance for the tumor to facilitate tumor-associated angiogenesis, e.g., the de novo formation of new blood vessels. In addition to established and key angiogenic factors, such as vascular endothelial growth factor, chemokines, a superfamily of cytokine-like proteins that bind to seven transmembrane-spanning G-protein-coupled receptors, have been associated with angiogenesis under homeostatic conditions. Chemokines were initially identified as key factors that control the directional migration of leukocytes, stem cells and cancer cells in vitro and which critically regulate their trafficking in vivo . Recently their role in establishing a favorable microenvironment for tumor-associated angiogenesis, a process that requires complex bidirectional interactions of the tumor and associated vessels, has been the focus of research. Chemokine-promoted angiogenesis not only facilitates tumor growth by supplying nutrients and oxygen but it is also a prerequisite to tumor metastasis. Hence, the pharmacologic control of tumor angiogenesis presents a promising strategy for novel anticancer therapeutics. Here, we discuss the current pathogenetic concepts of tumor-associated angiogenesis in the context of chemokines and their receptors and highlight promising therapeutic strategies.

The mammalian Ah receptor (AhR) is a ligand-activated transcription factor with multiple functions in adaptive metabolism, development and dioxin toxicity in a variety of organs and cell systems. Phenotypes observed following sustained activation by dioxin or in AhR-null mice suggest organ-dependent physiological functions. These functions are probably deregulated following exposure to dioxin. We focus on skin and liver to facilitate discussion of mechanisms linking phenotypes and AhR-modulated genotypes. After a brief summary of currently discussed AhR ligand candidates, two groups of direct AhR target genes/proteins and associated functions are highlighted: (i) xenobiotic-metabolizing enzymes which are also involved in homeostasis of endogenous ligands and (ii) proteins controlling cell proliferation/apoptosis, differentiation and inflammation. Homeostatic feedback loops might not only include CYP1A1 but also Phase II enzymes such as UGT1A1 which controls the antioxidant AhR ligand bilirubin. The AhR is involved in extensive crosstalk with other transcription factors and multiple signaling pathways. Efforts elucidating the pathway toward identification of physiological functions of the AhR remain challenging and promising.

DEAD box proteins catalyze the ATP-dependent unwinding of double-stranded RNA (dsRNA). In addition, they facilitate protein displacement and remodeling of RNA or RNA/protein complexes. Their hallmark feature is local destabilization of RNA duplexes. Here, we summarize current data on the DEAD box protein mechanism and present a model for RNA unwinding that integrates recent data on the effect of ATP analogs and mutations on DEAD box protein activity. DEAD box proteins share a conserved helicase core with two flexibly linked RecA-like domains that contain all helicase signature motifs. Variable flanking regions contribute to substrate binding and modulate activity. In the presence of ATP and RNA, the helicase core adopts a compact, closed conformation with extensive interdomain contacts and high affinity for RNA. In the closed conformation, the RecA-like domains form a catalytic site for ATP hydrolysis and a continuous RNA binding site. A kink in the backbone of the bound RNA locally destabilizes the duplex. Rearrangement of this initial complex generates a hydrolysis- and unwinding-competent state. From this complex, the first RNA strand can dissociate. After ATP hydrolysis and phosphate release, the DEAD box protein returns to a low-affinity state for RNA. Dissociation of the second RNA strand and reopening of the cleft in the helicase core allow for further catalytic cycles.

4-Hydroxybutyrate CoA-transferases (4-HB-CoAT) takes part in the fermentation of 4-aminobutyrate to ammonia, acetate, and butyrate in anaerobic bacteria such as Clostridium aminobutyricum and Porphyromonas gingivalis or facultative anaerobic bacteria such as Shewanella oneidensis . Site-directed mutagenesis of the highly active enzyme has identified the catalytic glutamate residue as E238. Crystal structure of this enzyme has been determined at a resolution of 1.85 Å. The 438-amino acid residue polypeptide chain folds into two topologically similar domains with an open α/β-fold, which is also found in other CoAT family I and family II members. The data indicate that the members of CoAT families I and II are closely related; the latter only lacking the catalytic glutamate residue. A putative co-substrate binding site for the 4-HB-CoAT was identified, in which a 4-hydroxybutyrate molecule has been modeled. This site is also responsible for binding the acetyl group of acetyl-CoA or the succinyl group of succinyl-CoA in succinyl-CoA:3-oxoacid CoA-transferase from mammalian mitochondria. Mutations of relevant active site amino acid residues have been produced and their activities tested to corroborate the proposed structural model for substrate binding. 4-HB-CoAT from C. aminobutyricum represents the only functionally characterized 4-HB-CoAT present in the structural database.

Binding of angiotensin II (DRVYIHPF, AngII) to its AT 1 receptor can trigger a process known as tachyphylaxis (loss of receptor response owing to repeated agonist stimulation). We propose a two-state binding model for tachyphylaxis where the N-terminal Asp 1 and Arg 2 residues of the peptide are supposed to initially bind to the N-terminal segment (Arg 23 ) and to the EC-3 loop (Asp 281 ) of an AT 1 molecule, respectively (state 1). Sequentially, a disruption of the salt bond between the AngII Asp 1 β-carboxyl function and the receptor Arg 23 can occur with release of the peptide N-terminal segment, favoring the binding of the Arg 2 residue to the EC-3 loop (Asp 178,281 , state 2). In the present study, we expanded this investigation by assaying pharmacological properties of different AngII analogs in guinea-pig ileum bearing modifications at positions 1 and 2. Most of these peptides were weak agonists but many of them had the ability to induce tachyphylaxis. These findings support the two-state model for tachyphylaxis, but alternative mechanisms were revealed where state 1 was no longer needed, depending on the chemical structure of AngII residue 1. Otherwise, any modification of the wild type AngII Arg 2 residue was deleterious for the tachyphylaxis mechanism.

Cancer Osaka thyroid (Cot) is a proto-oncogenic kinase which belongs to the MAP3K family. A peptide-based substrate screening assay revealed that Cot has the ability to phosphorylate Polo-like kinase 1 (Plk1) at Ser137. Kinase assays with intact Plk1 and peptides surrounding Ser137 and Thr210 indicated further that Cot phosphorylates Ser137 but not Thr210. Additional support came from 3D peptide structure prediction and Cot-Plk1 interaction modeling. In vivo experiments demonstrated that wild type Cot, but not a kinase-dead mutant, has the ability to phosphorylate Ser137. Knockdown of Cot in Hela showed a reduction in the level of phosphorylation of Ser137. These results imply for the first time that Cot might be an upstream kinase of Plk1 and suggest a new mechanism for the regulation of the cellular function of Plk1.

Coagulation factor XIII (FXIII) is activated by thrombin and catalyses crosslinking between fibrin monomers thereby providing mechanical strength to the fibrin network. V34L is a common FXIII-A polymorphism found in the activation peptide. FXIII-A V34L is activated faster by thrombin and provides formation of a tighter clot at fibrinogen concentrations in the low end of the physiological range. FXIII-A variants with potentially increased activation rates were generated. Introduction of an optimal thrombin cleavage site, V34L+V35T, increased the activation rate 7.6-fold and facilitated the formation of a fibrin network more resistant to fibrinolysis than obtained with wt FXIII-A. In contrast, introduction of fragments of fibrinopeptide A into the activation peptide resulted in severely impaired activation rates.

The cysteine protease CMS2MS2 from Carica candamarcensis latex has been shown to enhance proliferation of L929 fibroblast and to activate the extracellular signal-regulated protein kinase (ERK). In experiments with CMS2MS2 irreversibly inhibited by E-64, the proliferative effect on fibroblasts remains unaffected. ERK phosphorylation mediated by CMS2MS2 was abolished in the presence of PD 98059 or U0126, both MAPK cascade inhibitors. In addition, these inhibitors suppress the mitogenic activity of intact CMS2MS2 or CMS2MS2-E-64. Furthermore, ERK phosphorylation and the mitogenic effect are partially suppressed by a phospholipase C (PLC) inhibitor. These data suggest that the mitogenic effect of CMS2MS2 on fibroblasts is independent of its proteolytic activity, requires ERK phosphorylation, and involves activation of PLC.

The EGF-related protein EFEMP1 (EGF-containing fibulin-like extracellular matrix protein 1) has been shown to promote tumor growth in human adenocarcinoma. To understand the mechanism of this action, the signal transduction activated upon treatment with this protein has been investigated. We show that EFEMP1 binds EGF receptor (EGFR) in a competitive manner relative to epidermal growth factor (EGF), implicating that EFEMP1 and EGF share the same or adjacent binding sites on the EGFR. Treatment of pancreatic carcinoma cells with purified EFEMP1 activates autophosphorylation of EGFR at the positions Tyr-992 and Tyr-1068, but not at the position Tyr-1048. This signal is further transduced to phosphorylation of Akt at position Thr-308 and p44/p42 MAPK (mitogen-activated protein kinase) at positions Thr-202 and Tyr-204. These downstream phosphorylation events can be inhibited by treatment with the EGFR kinase inhibitor PD 153035. The observed signal transduction upon treatment with EFEMP1 can contribute to the enhancement of tumor growth shown in pancreatic carcinoma cells overexpressing EFEMP1 .

The immune system can be stimulated by microbial molecules as well as by endogenously derived danger/alarm signals of host origin. Using the lepidopteran model insect Galleria mellonella , we recently discovered that fragments of collagen IV, resulting from hydrolysis by microbial metalloproteinases, represent danger/alarm signals in insects. Here, we characterized immune-stimulatory peptides generated by thermolysin-mediated degradation of collagen IV using nanospray ionization Fourier transform ion cyclotron resonance mass spectrometry (FTICR MS) after separation by nanoscale liquid chromatography (nanoLC). The combination of FTICR MS analysis and de novo peptide sequencing resulted in the identification of 38 specific collagen IV fragments of which several peptides included the integrin-binding motif RGD/E known from numerous mammalian immune-related proteins. Custom-synthesized peptides corresponding either to the presently identified collagen peptide GIRGEHyp or to a well-known integrin-binding RGD peptide (GRGDS) were injected into G. mellonella to determine their immune-stimulatory activities in vivo . Both peptides stimulated immune cells and systemically the expression of lysozyme and a specific inhibitor of microbial metalloproteinases. Further examination using specific MAP kinase inhibitors indicated that MEK/ERK and p38 are involved in RGD/E-mediated immune-signaling pathways, whereas JNK seems to play only a minor role.

Porphyromonas gingivalis , the major causative bacterium of periodontitis, contributes significantly to elevated proteolytic activity at periodontal pockets owing to the presence of both bacteria and host, predominantly neutrophil-derived, serine proteases. Normally the activity of the latter enzymes is tightly regulated by endogenous proteins, including elafin, a potent neutrophil elastase and proteinase 3 inhibitor released from epithelial cells at sites of inflammation. Here, we report that all three gingipains (HRgpA, RgpB, and Kgp) have the ability to degrade elafin, with RgpB being far more efficient than other gingipains. RgpB efficiently inactivates the inhibitory activity of elafin at subnanomolar concentrations through proteolysis limited to the Arg22-Cys23 peptide bond within the surface loop harboring the inhibitor active site. Notably, elafin resists inactivation by several Staphylococcus aureus -derived serine and cysteine proteases, confirming the high stability of this protein against proteolytic degradation. Therefore, we conclude that elafin inactivation by RgpB represents a specific pathogenic adaptation of P. gingivalis to disturb the protease-protease inhibitor balance in the infected gingival tissue. This contributes to enhanced degradation of host proteins and generation of a pool of peptides serving as nutrients for this asaccharolytic pathogen.

No abstract available

No abstract available

No abstract available

No abstract available