Matthias Engel

Glyceraldehyde-3-phosphate Dehydrogenase and Nm23-H1/Nucleoside Diphosphate Kinase A TWO OLD ENZYMES COMBINE FOR THE NOVEL Nm23 PROTEIN PHOSPHOTRANSFERASE FUNCTION

We have recently discovered an alternative function of the putative metastasis suppressor protein Nm23, which is identical to nucleoside diphosphate kinase, as a protein phosphotransferase in vitro. While purified native Nm23 protein did not phosphorylate other proteins, we could purify a Nm23-associated protein that activates the protein phosphotransferase function; it was identified as a glyceraldehyde-3-phosphate dehydrogenase (GAPDH) isoenzyme. Co-expression and purification of (His)6-tagged GAPDH in combination with either Nm23-H1 or Nm23-H2 in baculovirus-infected Sf9 cells showed that only Nm23-H1, but not Nm23-H2, forms a stable complex with GAPDH. Protein phosphotransferase activity was confirmed for the recombinant GAPDH·Nm23-H1 complex but not for either of the enzymes alone, nor was this activity observed after simple mixing of the purified proteinsin vitro. The molecular mass of the highly purified recombinant GAPDH·Nm23-H1 complex suggests that a dimer of GAPDH interacts with a dimer of Nm23-H1. In contrast to the complex with GAPDH, co-expression of Nm23-H1 with antioxidant protein (MER-5) or creatine kinase did not activate the protein phosphotransferase function, indicating that this activation may specifically require GAPDH as a binding partner.

TRPA1 and Substance P Mediate Colitis in Mice

BACKGROUND & AIMS The neuropeptides calcitonin gene-related peptide (CGRP) and substance P, and calcium channels, which control their release from extrinsic sensory neurons, have important roles in experimental colitis. We investigated the mechanisms of colitis in 2 different models, the involvement of the irritant receptor transient receptor potential of the ankyrin type-1 (TRPA1), and the effects of CGRP and substance P. METHODS We used calcium-imaging, patch-clamp, and neuropeptide-release assays to evaluate the effects of 2,4,6-trinitrobenzene-sulfonic-acid (TNBS) and dextran-sulfate-sodium-salt on neurons. Colitis was induced in wild-type, knockout, and desensitized mice. RESULTS TNBS induced TRPA1-dependent release of colonic substance P and CGRP, influx of Ca2+, and sustained ionic inward currents in colonic sensory neurons and transfected HEK293t cells. Analysis of mutant forms of TRPA1 revealed that TNBS bound covalently to cysteine (and lysine) residues in the cytoplasmic N-terminus. A stable sulfinic acid transformation of the cysteine-SH group, shown by mass spectrometry, might contribute to sustained sensitization of TRPA1. Mice with colitis had increased colonic neuropeptide release, mediated by TRPA1. Endogenous products of inflammatory lipid peroxidation also induced TRPA1-dependent release of colonic neuropeptides; levels of 4-hydroxy-trans-2-nonenal increased in each model of colitis. Colitis induction by TNBS or dextran-sulfate-sodium-salt was inhibited or reduced in TRPA1-/- mice and by 2-(1,3-dimethyl-2,6-dioxo-1,2,3,6-tetrahydro-7H-purin-7-yl)-N-(4-isopro-pylphenyl)-acetamide, a pharmacologic inhibitor of TRPA1. Substance P had a proinflammatory effect that was dominant over CGRP, based on studies of knockout mice. Ablation of extrinsic sensory neurons prevented or attenuated TNBS-induced release of neuropeptides and both forms of colitis. CONCLUSIONS Neuroimmune interactions control intestinal inflammation. Activation and sensitization of TRPA1 and release of substance P induce and maintain colitis in mice.

Structure and allosteric effects of low-molecular-weight activators on the protein kinase PDK1.

Protein phosphorylation transduces a large set of intracellular signals. One mechanism by which phosphorylation mediates signal transduction is by prompting conformational changes in the target protein or interacting proteins. Previous work described an allosteric site mediating phosphorylation-dependent activation of AGC kinases. The AGC kinase PDK1 is activated by the docking of a phosphorylated motif from substrates. Here we present the crystallography of PDK1 bound to a rationally developed low-molecular-weight activator and describe the conformational changes induced by small compounds in the crystal and in solution using a fluorescence-based assay and deuterium exchange experiments. Our results indicate that the binding of the compound produces local changes at the target site, the PIF binding pocket, and also allosteric changes at the ATP binding site and the activation loop. Altogether, we present molecular details of the allosteric changes induced by small compounds that trigger the activation of PDK1 through mimicry of phosphorylation-dependent conformational changes.

Allosteric activation of the protein kinase PDK1 with low molecular weight compounds

Organisms rely heavily on protein phosphorylation to transduce intracellular signals. The phosphorylation of a protein often induces conformational changes, which are responsible for triggering downstream cellular events. Protein kinases are themselves frequently regulated by phosphorylation. Recently, we and others proposed the molecular mechanism by which phosphorylation at a hydrophobic motif (HM) regulates the conformation and activity of many members of the AGC group of protein kinases. Here we have developed specific, low molecular weight compounds, which target the HM/PIF‐pocket and have the ability to allosterically activate phosphoinositide‐dependent protein kinase 1 (PDK1) by modulating the phosphorylation‐dependent conformational transition. The mechanism of action of these compounds was characterized by mutagenesis of PDK1, synthesis of compound analogs, interaction‐displacement studies and isothermal titration calorimetry experiments. Our results raise the possibility of developing drugs that target the AGC kinases via a novel mode of action and may inspire future rational development of compounds with the ability to modulate phosphorylation‐dependent conformational transitions in other proteins.

Ciguatoxins activate specific cold pain pathways to elicit burning pain from cooling

Ciguatoxins are sodium channel activator toxins that cause ciguatera, the most common form of ichthyosarcotoxism, which presents with peripheral sensory disturbances, including the pathognomonic symptom of cold allodynia which is characterized by intense stabbing and burning pain in response to mild cooling. We show that intraplantar injection of P‐CTX‐1 elicits cold allodynia in mice by targeting specific unmyelinated and myelinated primary sensory neurons. These include both tetrodotoxin‐resistant, TRPA1‐expressing peptidergic C‐fibres and tetrodotoxin‐sensitive A‐fibres. P‐CTX‐1 does not directly open heterologously expressed TRPA1, but when co‐expressed with Nav channels, sodium channel activation by P‐CTX‐1 is sufficient to drive TRPA1‐dependent calcium influx that is responsible for the development of cold allodynia, as evidenced by a large reduction of excitatory effect of P‐CTX‐1 on TRPA1‐deficient nociceptive C‐fibres and of ciguatoxin‐induced cold allodynia in TRPA1‐null mutant mice. Functional MRI studies revealed that ciguatoxin‐induced cold allodynia enhanced the BOLD (Blood Oxygenation Level Dependent) signal, an effect that was blunted in TRPA1‐deficient mice, confirming an important role for TRPA1 in the pathogenesis of cold allodynia.

Expression of calpain I messenger rna in human renal cell carcinoma : Correlation with lymph node metastasis and histological type

Calpain, also named CANP (for calcium‐activated neutral protease), is an intracellular cytoplasmatic non‐lysosomal cysteine endopeptidase that requires calcium ions for activity. Many substrates of the calpain isoenzymes, such as the transcription factors c‐Fos and c‐Jun, the tumor supressor protein p53, protein kinase C, pp60c‐src and the adhesion molecule integrin, have been implicated in the pathogenesis of different human tumors, suggesting an important role of the calpains in malignant diseases. We now report differential expression of the calpain I gene (CL 1) in a variety of tumors, extending our study to a larger series of renal cell carcinomas. Using Northern‐blot analysis, we studied calpain I expression in 30 renal cell carcinomas as compared with matched healthy tissues. Tumor samples were classified according to their histological type: 21 clear cell carcinomas, 4 chromophobe carcinomas, 3 papillary carcinomas and 2 oncocytomas. In renal tumor samples, calpain I gene mRNA was expressed at highly variable levels, significantly depending on the different histological types. Moreover, there was a correlation of higher calpain I expression with increased malignancy: within the clear cell carcinoma subset, tumor samples with advanced nodal status (N1 and N2) showed a significantly higher calpain I expression than tumors without metastasis to regional lymph nodes. Our data suggest an important role of calpain isoenzymes in carcinogenesis and tumor progression. Int. J. Cancer (Pred. Oncol.) 84:6–9, 1999.

Role of sensory neurons in colitis: increasing evidence for a neuroimmune link in the gut

Growing evidence suggests a crucial involvement of extrinsic sensory neurons in the aberrant immune response in colitis. Activation of sensory neurons is accompanied by a release of the neuropeptides calcitonin gene-related peptide (CGRP) and substance P (SP), which induce neurogenic inflammation characterized by vasodilatation, plasma extravasation, and leukocyte migration. Although the role of these neuropeptides in experimental colitis and human inflammatory bowel disease (IBD) remains controversial, numerous data indicate a functional role for sensory neurons. In fact, chemical desensitization or surgical denervation of sensory nerves were shown to attenuate experimental colitis. Furthermore, pharmacological blockade of the neurokinin-1 (NK1) receptor was demonstrated to be efficient in chemically induced mouse models of colitis, and, intriguingly, also in immune-mediated models of colitis (T-cell transfer colitis). Finally, the genetic deletion or pharmacological blockade of receptor channels such as TRPV1 and TRPA1 on nociceptive sensory neurons was also demonstrated to be effective in treating experimental colitis, supposedly by inhibiting neuropeptide release. In summary, we are only beginning to understand the mechanisms of how sensory neurons modulate immune cellular actions. These findings highlight a new role of sensory neurons in chronic intestinal inflammation and suggest new avenues for therapy of IBD.

The proximodistal aggravation of colitis depends on substance P released from TRPV1-expressing sensory neurons

BackgroundTransient receptor potential vanilloid type-1 (TRPV1)-expressing sensory neurons release neuropeptides such as substance P (SP) and calcitonin gene-related peptide (CGRP), which play a crucial role in the pathomechanism of experimental colitis. We investigated whether innervation density and neuropeptide release were responsible for the proximodistal aggravation of murine dextran-sulfate-sodium-salt (DSS) colitis.MethodsWhole mount TRPV1/CGRP immunostained mouse colon preparations were semiquantitatively analyzed. TRPV1 activation by capsaicin and acidic solution (pH 5.1) induced colonic CGRP/SP release, measured by EIA. Single cell quantitative PCR was employed to measure TRPV1 expression levels in DiI-labeled colonic dorsal root ganglion (DRG) neurons. The proximodistal gradient of DSS colitis severity was investigated in WT, CGRP−/−, SP−/−, and resiniferatoxin (RTX)-desensitized mice, employing mouse endoscopy, histology, and body weight measurement.ResultsTRPV1/CGRP-positive nerve fiber density was increased in the distal colon wall. CGRP/SP release induced by TRPV1 activation from the distal colon was greater than that from the proximal colon. This gradient further increased in colitis. TRPV1 gene expression increased in colonic DRGs projecting to the distal, compared to that in colonic DRGs projecting to the proximal colon, and was further enhanced during colitis. In contrast to WT and CGRP−/− mice, SP−/− and RTX-desensitized mice showed amelioration of DSS colitis accompanied by a loss of the proximodistal gradient of inflammation.ConclusionsThe spatial correlation among increased colonic innervation density, TRPV1 receptor expression, stimulated SP release, and colitis severity suggested that TRPV1/SP-expressing sensory neurons should be considered as a therapeutic target in human ulcerative colitis.

3,5-Diphenylpent-2-enoic acids as allosteric activators of the protein kinase PDK1: structure-activity relationships and thermodynamic characterization of binding as paradigms for PIF-binding pocket-targeting compounds.

The modulation of protein kinase activities by low molecular weight compounds is a major goal of current pharmaceutical developments. In this line, important efforts are directed to the development of drugs targeting the conserved ATP binding site. However, there is very little experience on targeting allosteric, regulatory sites, different from the ATP binding site, in protein kinases. Here we describe the synthesis, cell-free activation potency, and calorimetric binding analysis of 3,5-diphenylpent-2-enoic acids and derivatives as allosteric modulators of the phosphoinositide-dependent kinase-1 (PDK1) catalytic activity. Our SAR results combined with thermodynamic binding analyses revealed both favorable binding enthalpy and entropy and confirmed the PIF-binding pocket of PDK1 as a druggable site. In conclusion, we defined the minimal structural requirements for compounds to bind to the PIF-binding pocket and to act as allosteric modulators and identified two new lead structures (12Z and 13Z) with predominating binding enthalpy.

Allosteric Regulation of Protein Kinase PKCζ by the N-Terminal C1 Domain and Small Compounds to the PIF-Pocket

Protein kinases are key mediators of cellular signaling, and therefore, their activities are tightly controlled. AGC kinases are regulated by phosphorylation and by N- and C-terminal regions. Here, we studied the molecular mechanism of inhibition of atypical PKCζ and found that the inhibition by the N-terminal region cannot be explained by a simple pseudosubstrate inhibitory mechanism. Notably, we found that the C1 domain allosterically inhibits PKCζ activity and verified an allosteric communication between the PIF-pocket of atypical PKCs and the binding site of the C1 domain. Finally, we developed low-molecular-weight compounds that bind to the PIF-pocket and allosterically inhibit PKCζ activity. This work establishes a central role for the PIF-pocket on the regulation of PKCζ and allows us to envisage development of drugs targeting the PIF-pocket that can either activate or inhibit AGC kinases.