Both cyclooxygenase-1 and -2 are expressed in the spinal cord, and the spinal COX product prostaglandin E(2) (PGE(2)) contributes to the generation of central sensitization upon peripheral inflammation. Vice versa spinal COX inhibition is considered an important mechanism of antihyperalgesic pain treatment. Recently, however, COX-2 was shown to be also involved in the metabolism of endocannabinoids. Because endocannabinoids can have analgesic actions it is conceivable that inhibition of spinal COX produces analgesia not only by inhibition of PG synthesis but also by inhibition of endocannabinoid breakdown. In the present study, we recorded from spinal cord neurons with input from the inflamed knee joint and we measured the spinal release of PGE(2) and the endocannabinoid 2-arachidonoyl glycerol (2-AG) in vivo, using the same stimulation procedures. COX inhibitors were applied spinally. Selective COX-1, selective COX-2 and non-selective COX inhibitors attenuated the generation of spinal hyperexcitability when applied before and during development of inflammation but, when inflammation and spinal hyperexcitability were established, only selective COX-2 inhibitors reversed spinal hyperexcitability. During established inflammation all COX inhibitors reduced release of spinal PGE(2) almost equally but only the COX-2 inhibitor prevented breakdown of 2-AG. The reversal of spinal hyperexcitability by COX-2 inhibitors was prevented or partially reversed by AM-251, an antagonist at the cannabinoid-1 receptor. We conclude that inhibition of spinal COX-2 not only reduces PG production but also endocannabinoid breakdown and provide evidence that reversal of inflammation-evoked spinal hyperexcitability by COX-2 inhibitors is more related to endocannabinoidergic mechanisms than to inhibition of spinal PG synthesis.

A versatile, multidimensional, and non-denaturing proteome separation procedure using microplate technology is presented, yielding a digitized image of proteome composition. In the first dimension, the sample under study is separated into 96 fractions by size exclusion chromatography (SEC). In the second dimension, the fractions of the first dimension are transferred by the liquid-handling device CyBi-Well (CyBio AG, Jena, Germany) to 96 parallel anion exchange chromatography columns. In this way the proteins are conserved in their native states and are distributed in 2400 liquid fractions with high recovery rates and sufficient reproducibility. The resulting fractions are subjected to protein quantitation and identification. Spectrophotometrical and immunological methods and enzyme activity measurements are used for quantitation. To identify proteins, the fractions are subjected to MALDI-MS, and their tryptic digests to both MALDI- and LC-ESI-MS/MS. All preparation steps except the first are applied in parallel to sets of multiples of 96 samples. The procedure may be refined by adding more separation steps and may be adapted to various protein amounts and to various proteomes. Moreover, the method offers the opportunity to investigate functional protein complexes. The method was applied to separate the normal human serum proteome. Within 255 fractions exhibiting the highest protein concentrations, 742 proteins were identified by LC-ESI-MS/MS peptide sequence tags.

Cathepsin S, a lysosomal cysteine protease, is synthesized as inactive precursor. It is activated in the lysosomes by a proteolytic cleavage of the propeptide. HEK 293-cells which do not express cathepsin S were transfected with cDNA of either wild type human procathepsin S or a mutant procathepsin S in which Asn of the only glycosylation site in the proregion was replaced by Gln. The cells expressed glycosylated and non-glycosylated procathepsin S, respectively. Large amounts of the precursors were secreted into the culture media by both transfectants. Secreted wild type procathepsin S contained Man-6-phosphate in the oligosaccharide chain. Wild type procathepsin S was activated in the cells but no maturation occurred in the culture media. In vitro processing of glycosylated as well as of non-glycosylated procathepsin S gave fully active enzymes thus indicating that the oligosaccharide chain was not necessary for proper folding. A reuptake of the glycosylated and non-glycosylated procathepsin S by HEK 293-cells could be observed. Small amounts of mature cathepsin S were detected in the lysosomes of the mutant transfectants. Subcellular fractionation showed non-glycosylated procathepsin S in the membrane fraction. Non-glycosylated procathepsin S was bound to the plasma membrane at 2 degrees C, suggesting an additional sorting motif in the cathepsin S molecule besides the Man-6-phosphate residue.

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Cathepsin L-like cysteine proteinases contain an evolutionarily highly conserved a-helical motif in the proregion. This is called the ER(F/W)N(I/V)N motif according to the conserved amino acids along one side of the helix. We studied the function of this motif using site-directed mutagenesis experiments of human procathepsin S. We replaced each of these amino acids with alanine and constructed deletion mutants lacking parts of the helix. All mutants were expressed in HEK 293 cells, but only one, W52A, was not processed to mature cathepsin S, nor was it phosphorylated or secreted into the culture medium. W52 is part of the hydrophobic core in the propeptide region of cathepsin S comprising two additional tryptophan residues, W28 and W31, also conserved among cathepsin L-like cysteine peptidases. Replacement of the latter with alanine led to consequences similar to those with the W52A mutation. Recombinant propeptides containing mutations of one of the three tryptophan residues were three orders of magnitude less effective as inhibitors of mature cathepsin S than the wild-type propeptide. The results point to a dominant role of the respective hydrophobic stack in the proper folding, transport and maturation of procathepsin S and related cathepsin L-like cysteine proteinases.

Two processes, synthesis and degradation, contribute to the intracellular concentration of a protein. As most malignant tumors or tumor cell lines show elevated levels of proteinases, we studied the half-life of a cysteine proteinase, procathepsin S, in order to determine whether tumor cells can regulate their cathepsin concentration via changing the degradation rate of the enzyme.The following procathepsin S species were examined: wild-type procathepsin S in macrophages, recombinant procathepsin S in human embryonic kidney cells (HEK 293 cells), recombinant nonglycosylated procathepsin S in HEK 293 cells, wild-type procathepsin S in the established nonsmall cell lung carcinoma cell line 97TM1.The half-lives of both wild-type procathepsins S expressed in macrophages and in HEK 293 cells were 1 h, whereas that of procathepsin S in the tumor cell line was 2 h. Nonglycosylated procathepsin S was not processed. The degradation of mature cathepsin S proceeded with a half-life of 16±18 h. All cell lines studied secreted substantial amounts of procathepsin S into the culture medium. No further maturation of secreted procathepsin S has been observed in the culture medium. We suggest a disturbed sorting mechanism in tumor cells.Keywords: procathepsin S; degradation; secretion; processing; tumor cells. Lysosomal proteins show long average half-lives (t12 ) of several days up to 2 weeks [1±3] although the accumulation of proteolytic enzymes in the lysosomes and the acid pH within this organelle create an appropriate environment for rapid protein degradation. The molecular properties rendering the lysosomal proteins resistant in vivo to the proteolytic attack are still unclear. Glycosylation has been reported to stabilize lysosomal proteins against rapid degradation [4], and the inhibition of hydrolytic enzymes by glycosaminoglycans within the lysosomes may also contribute to this stability [5]. In contrast, isolated cathepsins are rapidly inactivated and degraded in vitro under weakly acidic conditions [6] at which these enzymes are most active. It remains to be further elucidated what makes lysosomal proteins, including the cathepsins, relatively long-lived.The maturation of procathepsins is a rapid process compared to the long life of the mature cathepsins, lasting 1 h or less for cathepsin D [1,4,7] and cysteine proteinases [8±12], as well as for other lysosomal proteins [13]. Reports dealing with the t12 -values of individual mature lysosomal cathepsins are sparse [1±3,14]. The biosynthesis and the maturation of inactive precursor molecules contribute to the supply of active lysosomal enzymes in the cell. Loss of active enzymes, either by secretion or by degradation, is the other determinant of lysosomal enzyme concentration. The t1 2 of an enzyme describes the turnover rate and, thus, it allows the calculation of the time scale at which the turnover rate can be regulated.Many malignant tumors and established tumor cell lines are characterized by high levels of proteolytic enzymes, including lysosomal cysteine proteina...

Cathepsin L-like cysteine proteinases contain an evolutionarily highly conserved a-helical motif in the proregion. This is called the ER(F/W)N(I/V)N motif according to the conserved amino acids along one side of the helix. We studied the function of this motif using site-directed mutagenesis experiments of human procathepsin S. We replaced each of these amino acids with alanine and constructed deletion mutants lacking parts of the helix. All mutants were expressed in HEK 293 cells, but only one, W52A, was not processed to mature cathepsin S, nor was it phosphorylated or secreted into the culture medium. W52 is part of the hydrophobic core in the propeptide region of cathepsin S comprising two additional tryptophan residues, W28 and W31, also conserved among cathepsin L-like cysteine peptidases. Replacement of the latter with alanine led to consequences similar to those with the W52A mutation. Recombinant propeptides containing mutations of one of the three tryptophan residues were three orders of magnitude less effective as inhibitors of mature cathepsin S than the wild-type propeptide. The results point to a dominant role of the respective hydrophobic stack in the proper folding, transport and maturation of procathepsin S and related cathepsin L-like cysteine proteinases.