The alpha1/2 helical backbone of the prodomains defines the intrinsic inhibitory specificity in the cathepsin L-like cysteine protease subfamily.

Proregions of papain-like cysteine proteases are potent and often highly selective inhibitors of their parental enzymes. The molecular basis of their selectivity is poorly understood. For two closely related members of the cathepsin L-like subfamily we established strong selectivity differences. The propeptide of cathepsin S was observed to inhibit cathepsin L with a K(i) of 0.08 nM, yet cathepsin L propeptide inhibited cathepsin S only poorly. To identify the respective structural correlates we engineered chimeric propeptides and compared their inhibitory specificity with the wild-types. Specificity resided in the N-terminal part, strongly suggesting that the backbone of the prodomain was the underlying structure.

[Exclusion of receptive speech disorders with the ADOS (Autism Diagnostic Observation Schedule)]. / Ausschluss rezeptiver Sprachstörungen mittels des ADOS (Autism Diagnostic Observation Schedule).

OBJECTIVE: The purpose of our pilot study was to assess the reliability and diagnostic validity of the Autism Diagnostic Observation Schedule (ADOS). The usefulness of the schedule in the differentiation between children with autism and children with a severe specific receptive language disorder is examined. METHOD: Eight boys with early infantile autism and eight age- and IQ-matched boys with a specific receptive language disorder were examined with the ADOS. The reliability of the instrument was assessed using the ratings of eight pairs of raters. The agreement between diagnostic classification based on the ADOS ICD-10 algorithm and the independent clinical psychiatric diagnosis of two experts was used as the measure of validity. RESULTS: The reliability of the different ADOS items proved to be good among experienced raters. Various ADOS items clearly discriminate both groups. Using the ADOS ICD-10 algorithm, the clinical diagnosis of infantile autism could be confirmed for five of the eight children in this group. None of the children with the clinical diagnosis of a receptive language disorder was identified as autistic according to the algorithm. CONCLUSIONS: In the hands of experienced raters the ADOS is a reliable diagnostic instrument. It can support the differentiation between autism and specific receptive language disorder, but additional parent information is needed to confirm the diagnosis.

A guanylyl cyclase from Paramecium with 22 transmembrane spans. Expression of the catalytic domains and formation of chimeras with the catalytic domains of mammalian adenylyl cyclases.

Paramecium has a 280-kDa guanylyl cyclase. The N terminus resembles a P-type ATPase, and the C terminus is a guanylyl cyclase with the membrane topology of canonical mammalian adenylyl cyclases, yet with the cytosolic loops, C1 and C2, inverted compared with the mammalian order. We expressed in Escherichia coli the cytoplasmic domains of the protozoan guanylyl cyclase, independently and linked by a peptide, as soluble proteins. The His(6)-tagged proteins were enriched by affinity chromatography and analyzed by immunoblotting. Guanylyl cyclase activity was reconstituted upon mixing of the recombinant C1a- and C2-positioned domains and in a linked C1a-C2 construct. Adenylyl cyclase activity was minimal. The nucleotide substrate specificity was switched from GTP to ATP upon mutation of the substrate defining amino acids Glu(1681) and Ser(1748) in the C1-positioned domain to the adenylyl cyclase specific amino acids Lys and Asp. Using the C2 domains of mammalian adenylyl cyclases type II or IX and the C2-positioned domain from the Paramecium guanylyl cyclase we reconstituted a soluble, all C2 adenylyl cyclase. All enzymes containing protozoan domains were not affected by Galpha(s)/GTP or forskolin, and P site inhibitors were only slightly effective.

Adenylyl cyclase Rv1625c of Mycobacterium tuberculosis: a progenitor of mammalian adenylyl cyclases.

The gene Rv1625c from Mycobacterium tuberculosis encodes a membrane-anchored adenylyl cyclase corresponding to exactly one-half of a mammalian adenylyl cyclase. An engineered, soluble form of Rv1625c was expressed in Escherichia coli. It formed a homodimeric cyclase with two catalytic centers. Amino acid mutations predicted to affect catalysis resulted in inactive monomers. A single catalytic center with wild-type activity could be reconstituted from mutated monomers in stringent analogy to the mammalian heterodimeric cyclase structure. The proposed existence of supramolecular adenylyl cyclase complexes was established by reconstitution from peptide-linked, mutation-inactivated homodimers resulting in pseudo-trimeric and -tetrameric complexes. The mycobacterial holoenzyme was expressed successfully in E.coli and mammalian HEK293 cells, i.e. its membrane targeting sequence was compatible with the bacterial and eukaryotic machinery for processing and membrane insertion. The membrane-anchored mycobacterial cyclase expressed in E.coli was purified to homogeneity as a first step toward the complete structural elucidation of this important protein. As the closest progenitor of the mammalian adenylyl cyclase family to date, the mycobacterial cyclase probably was spread by horizontal gene transfer.

Cathepsin L is an intracellular and extracellular protease in Paramecium tetraurelia. Purification, cloning, sequencing and specific inhibition by its expressed propeptide.

The ciliate Paramecium tetraurelia secretes large amounts of a cysteine protease into the growth medium, presumably for extracellular food digestion. Two endoprotease isozymes (30 and 33 kDa on SDS/PAGE, respectively), both present in cell homogenates and in spent growth medium, were purified to homogeneity. Peptide sequence analysis revealed that these isozymes share identities at the amino acid level but are probably differently processed. Enzymatic characterization of the isolated proteases and sequencing of the cloned cDNA demonstrated that the enzymes belong to the cathepsin-L protease subfamily. Although the identity with mammalian and other protozoan L cathepsins was only around 30%, all important signature sequences for cathepsin L in the preproregion as well as in the catalyst of the enzyme were fully retained. The cDNA of this cysteine protease codes for a preproregion of 108 amino acids. The putative proregion of 86 amino acids which contained the characteristic conserved ERFNIN motif, was fused with a His6 tag, expressed in Escherichia coli, and purified. Both cathepsin L isozymes of Paramecium tetraurelia were inhibited by their cognate propeptide in the nanomolar concentration range. All other cysteine proteases tested (papain and mammalian cathepsin B, G and H) were unaffected by the propeptide up to 10 microM.