Thilo Kähne

A degradation-sensitive anionic trypsinogen (PRSS2) variant protects against chronic pancreatitis

Chronic pancreatitis is a common inflammatory disease of the pancreas. Mutations in the genes encoding cationic trypsinogen (PRSS1) and the pancreatic secretory trypsin inhibitor (SPINK1) are associated with chronic pancreatitis. Because increased proteolytic activity owing to mutated PRSS1 enhances the risk for chronic pancreatitis, mutations in the gene encoding anionic trypsinogen (PRSS2) may also predispose to disease. Here we analyzed PRSS2 in individuals with chronic pancreatitis and controls and found, to our surprise, that a variant of codon 191 (G191R) is overrepresented in control subjects: G191R was present in 220/6,459 (3.4%) controls but in only 32/2,466 (1.3%) affected individuals (odds ratio 0.37; P = 1.1 × 10−8). Upon activation by enterokinase or trypsin, purified recombinant G191R protein showed a complete loss of trypsin activity owing to the introduction of a new tryptic cleavage site that renders the enzyme hypersensitive to autocatalytic proteolysis. In conclusion, the G191R variant of PRSS2 mitigates intrapancreatic trypsin activity and thereby protects against chronic pancreatitis.

Encoding and Transducing the Synaptic or Extrasynaptic Origin of NMDA Receptor Signals to the Nucleus

The activation of N-methyl-D-aspartate-receptors (NMDARs) in synapses provides plasticity and cell survival signals, whereas NMDARs residing in the neuronal membrane outside synapses trigger neurodegeneration. At present, it is unclear how these opposing signals are transduced to and discriminated by the nucleus. In this study, we demonstrate that Jacob is a protein messenger that encodes the origin of synaptic versus extrasynaptic NMDAR signals and delivers them to the nucleus. Exclusively synaptic, but not extrasynaptic, NMDAR activation induces phosphorylation of Jacob at serine-180 by ERK1/2. Long-distance trafficking of Jacob from synaptic, but not extrasynaptic, sites depends on ERK activity, and association with fragments of the intermediate filament α-internexin hinders dephosphorylation of the Jacob/ERK complex during nuclear transit. In the nucleus, the phosphorylation state of Jacob determines whether it induces cell death or promotes cell survival and enhances synaptic plasticity.

Posttranscriptional Control of Renin Synthesis: Identification of Proteins Interacting With Renin mRNA 3′-Untranslated Region

Abstract— Stabilization and correct localization of mRNA are important features of renin synthesis. To elucidate the molecular basis of cAMP-mediated posttranscriptional control via mRNA stabilization, we analyzed the interaction of human preprorenin (hREN) mRNA 3′-untranslated region (3′-UTR) with proteins of renin synthesizing Calu-6 cells and investigated their functional impact on messenger integrity. To identify hREN mRNA binding proteins, electrophoretic mobility shift assays, UV cross-linking and RNA-affinity chromatography with subsequent matrix-assisted laser desorption/ionization time-of-flight mass spectrometry were performed. The following six proteins were unambiguously identified as hREN mRNA 3′-UTR binding proteins: hnRNP E1 (synonyms &agr;-CP or PCBP), hnRNP K, dynamin, nucleolin, YB-1, and MINT-homologous protein. All proteins contain various RNA binding motifs, and most have been described in the context of mRNA binding and mRNA stabilization. Four proteins for which antibodies were available were verified by immunological techniques (dynamin, nucleolin, hnRNP E1, and YB-1). Forskolin, an activator of cAMP synthesis, considerably stimulates renin synthesis via inhibition of REN mRNA decay. Functionally, this cAMP-based mRNA stabilization is accompanied by a 3- to 6-fold upregulation of REN mRNA binding proteins. RNase degradation assays confirm that 3′-UTR binding proteins are able to protect and stabilize REN mRNA in vitro.

Proteolysis of AA Amyloid Fibril Proteins by Matrix Metalloproteinases-1, -2, and -3

We recently demonstrated the presence of matrix metalloproteinases (MMPs)-1, -2, and -3 in AA amyloid deposits, which lead us to speculate that MMPs may participate in amyloidogenesis by either processing the precursor protein, or by degrading the amyloid deposits. Here we investigated this theory by determining the ability of MMP-1, -2, and -3 to degrade human acute-phase serum amyloid A (SAA) and human AA amyloid fibril proteins (AFPs). The following in vitro degradation experiments were performed: using either recombinant MMP-1, -2, or -3 and SAA as a substrate; using either recombinant MMP-1, -2, or -3 and AFP as a substrate; and using THP-1 cells as the protease source and AFP as the substrate. All three MMPs were able to cleave SAA and AFP within the region spanning residues 51 to 57. The following cleavage sites were identified: at 57 to 58 for MMP-1; at 7 to 8 and 51 to 52 for MMP-2; at 7 to 8, 16 to 17, 23 to 24, 51 to 52, 55 to 56, 56 to 57, and 57 to 58 for MMP-3. Cell culture experiments showed that THP-1 cells were able to degrade AFPs. Degradation was significantly delayed after addition of a general metalloproteinase inhibitor (o-phenanthroline) to dextran sulfate-stimulated cells. This is the first study to show that human SAAs and AFPs are susceptible to proteolytic cleavage by MMPs. Immunocytochemistry and electron microscopy showed that degradation takes place in the pericellular or extracellular compartment.

Consequences of COP9 signalosome and 26S proteasome interaction

The COP9 signalosome (CSN) occurs in all eukaryotic cells. It is a regulatory particle of the ubiquitin (Ub)/26S proteasome system. The eight subunits of the CSN possess sequence homologies with the polypeptides of the 26S proteasome lid complex and just like the lid, the CSN consists of six subunits with PCI (proteasome, COP9 signalosome, initiation factor 3) domains and two components with MPN (Mpr‐Pad1‐N‐terminal) domains. Here we show that the CSN directly interacts with the 26S proteasome and competes with the lid, which has consequences for the peptidase activity of the 26S proteasome in vitro. Flag‐CSN2 was permanently expressed in mouse B8 fibroblasts and Flag pull‐down experiments revealed the formation of an intact Flag‐CSN complex, which is associated with the 26S proteasome. In addition, the Flag pull‐downs also precipitated cullins indicating the existence of super‐complexes consisting of the CSN, the 26S proteasome and cullin‐based Ub ligases. Permanent expression of a chimerical subunit (Flag‐CSN2‐Rpn6) consisting of the N‐terminal 343 amino acids of CSN2 and of the PCI domain of S9/Rpn6, the paralog of CSN2 in the lid complex, did not lead to the assembly of an intact complex showing that the PCI domain of CSN2 is important for complex formation. The consequence of permanent Flag‐CSN2 overexpression was de‐novo assembly of the CSN complex connected with an accelerated degradation of p53 and stabilization of c‐Jun in B8 cells. The possible role of super‐complexes composed of the CSN, the 26S proteasome and of Ub ligases in the regulation of protein stability is discussed.

Inhibitors of Dipeptidyl Peptidase IV (DP IV, CD26) Specifically Suppress Proliferation and Modulate Cytokine Production of Strongly CD26 Expressing U937 Cells

Various studies from different laboratories have shown that the membrane ectoenzyme dipeptidyl peptidase IV (DP IV, CD26) expressed in T and NK cells is involved in the regulation of DNA synthesis and cytokine production. In this paper, we performed a biochemical and functional characterization of dipeptidyl peptidase IV on the human histiocytic lymphoma cell line U937. Using U937 clones expressing low to high levels of membrane localized CD26, we found that the synthetic reversible inhibitors of DP IV, Lys-[Z(NO2)]-thiazolidide and Lys-[Z(NO2)]-piperidide, have different effects on all functions. In U937-H cells that strongly express high levels of CD26, DP IV inhibitors were shown to suppress DNA synthesis and production of IL-1 beta, but stimulate the secretion of the IL-1 receptor antagonist (IL-1RA) and of TNF-alpha. In contrast, both inhibitors did not influence the cytokine production and DNA synthesis in U937-L cells exhibiting low level CD26 expression. These data support the hypothesis that CD26 plays a crucial role in proliferation and cytokine production, not only in T cells, but also in other cell systems, and that enzymatic activity is essential for its function.

The role of dipeptidyl peptidase IV (DP IV) enzymatic activity in T cell activation and autoimmunity

Abstract Activated T lymphocytes express high levels of dipeptidyl peptidase IV (DP IV)/CD26. Recent studies support the notion that DP IV may play an important role in the regulation of differentiation and growth of T lymphocytes. This article gives a short overview on DP IV/CD26 expression and effects on immune cells in vitro and in vivo. A major focus of this review are clinical aspects of the function of CD26 on hematopoietic cells and the potential usage of synthetic DP IV inhibitors as therapeutics in inflammatory disorders.

Amino‐terminal truncation of procalcitonin, a marker for systemic bacterial infections, by dipeptidyl peptidase IV (DP IV)

Increased concentrations of procalcitonin (PCT) are found in the plasma of patients with thermal injury and in patients with sepsis and severe infection, making this molecule important as a diagnostic and prognostic marker in these diseases. Interestingly, only the truncated form of PCT, PCT(3‐116), is present in the plasma of these patients. The enzyme responsible for this truncation is unknown as yet. Here, using capillary zone electrophoresis, mass spectrometry and Edman sequence analysis, we demonstrate that dipeptidyl peptidase IV (DP IV, EC 3.4.14.5) is capable of catalyzing the hydrolysis of PCT(1‐116), releasing the N‐terminal dipeptide Ala‐Pro. We hypothesize that PCT(3‐116) is the result of the hydrolysis of PCT(1‐116) by soluble DP IV of the blood plasma or by DP IV expressed on the surface of cells.

Cathepsin L inactivates human trypsinogen whereas cathepsin L deletion reduces the severity of pancreatitis in mice

BACKGROUND & AIMS Acute pancreatitis is characterized by an activation cascade of digestive enzymes in the pancreas. The first of these, trypsinogen, can be converted to active trypsin by the peptidase cathepsin B (CTSB). We investigated whether cathepsin L (CTSL) can also process trypsinogen to active trypsin and has a role in pancreatitis. METHODS In CTSL-deficient (Ctsl(-/-)) mice, pancreatitis was induced by injection of cerulein or infusion of taurocholate into the pancreatic duct. Human tissue, pancreatic juice, mouse pancreatitis specimens, and recombinant enzymes were studied by enzyme assay, immunoblot, N-terminal sequencing, immunocytochemistry, and electron microscopy analyses. Isolated acini from Ctsl(-/-) and Ctsb(-/-) mice were studied. RESULTS CTSL was expressed in human and mouse pancreas, colocalized with trypsinogen in secretory vesicles and lysosomes, and secreted into pancreatic juice. Severity of pancreatitis was reduced in Ctsl(-/-) mice, whereas apoptosis and intrapancreatic trypsin activity were increased. CTSL-induced cleavage of trypsinogen occurred 3 amino acids toward the C-terminus from the CTSB activation site and resulted in a truncated, inactive form of trypsin and an elongated propeptide (trypsinogen activation peptide [TAP]). This elongated TAP was not detected by enzyme-linked immunosorbent assay (ELISA) but was effectively converted to an immunoreactive form by CTSB. Levels of TAP thus generated by CTSB were not associated with disease severity, although this is what the TAP-ELISA is used to determine in the clinic. CONCLUSIONS CTSL inactivates trypsinogen and counteracts the ability of CTSB to form active trypsin. In mouse models of pancreatitis, absence of CTSL induces apoptosis and reduces disease severity.

Subcellular localization of Grb2 by the adaptor protein Dok-3 restricts the intensity of Ca2+ signaling in B cells.

Spatial and temporal modulation of intracellular Ca2+ fluxes controls the cellular response of B lymphocytes to antigen stimulation. Herein, we identify the hematopoietic adaptor protein Dok‐3 (downstream of kinase‐3) as a key component of negative feedback regulation in Ca2+ signaling from the B‐cell antigen receptor. Dok‐3 localizes at the inner leaflet of the plasma membrane and is a major substrate for activated Src family kinase Lyn. Phosphorylated Dok‐3 inhibits antigen receptor‐induced Ca2+ elevation by recruiting cytosolic Grb2, which acts at this location as a negative regulator of Brutons tyrosine kinase. This leads to diminished activation of phospholipase C‐γ2 and reduced production of soluble inositol trisphosphate. Hence, the Dok‐3/Grb2 module is a membrane‐associated signaling organizer, which orchestrates the interaction efficiency of Ca2+‐mobilizing enzymes.