Crystal structure of cathepsin X: a flip-flop of the ring of His23 allows carboxy-monopeptidase and carboxy-dipeptidase activity of the protease.
(9/284)
BACKGROUND: Cathepsin X is a widespread, abundantly expressed papain-like mammalian lysosomal cysteine protease. It exhibits carboxy-monopeptidase as well as carboxy-dipeptidase activity and shares a similar activity profile with cathepsin B. The latter has been implicated in normal physiological events as well as in various pathological states such as rheumatoid arthritis, Alzheimer's disease and cancer progression. Thus the question is raised as to which of the two enzyme activities has actually been monitored. RESULTS: The crystal structure of human cathepsin X has been determined at 2.67 A resolution. The structure shares the common features of a papain-like enzyme fold, but with a unique active site. The most pronounced feature of the cathepsin X structure is the mini-loop that includes a short three-residue insertion protruding into the active site of the protease. The residue Tyr27 on one side of the loop forms the surface of the S1 substrate-binding site, and His23 on the other side modulates both carboxy-monopeptidase as well as carboxy-dipeptidase activity of the enzyme by binding the C-terminal carboxyl group of a substrate in two different sidechain conformations. CONCLUSIONS: The structure of cathepsin X exhibits a binding surface that will assist in the design of specific inhibitors of cathepsin X as well as of cathepsin B and thereby help to clarify the physiological roles of both proteases. (+info)
A new specialized cell-matrix interaction in actively resorbing osteoclasts.
(10/284)
We have identified a novel cell-matrix interaction in activated osteoclasts. Resorbing osteoclasts maintain a barrier adjacent to the bone surface that prevents the leakage of secreted protons and proteases from the resorption area. Using a series of fluorescent dyes of known molecular mass and different surface charge we established that negatively charged molecules with M(r )up to 10,000 rapidly accumulate underneath actively resorbing osteoclasts. Live cell imaging shows that staining could be detected underneath the osteoclasts as early as 30 seconds after the addition of the low molecular mass markers. We provide evidence that the actin cytoskeleton and the adhesion substrate in contact with the cells are critically involved in the maintenance of the sealing barrier. These data taken together suggest that the accumulation under resorbing osteoclasts is by diffusion rather than transcytotic delivery. Our results indicate that the net concentration of secreted and resorbed components is a balance between generation rate and limited diffusion rather than the presence of an impermeable barrier as previously suggested. This dynamic osteoclast sealing zone may, thus, provide the mechanism by which osteoclast migration and resorption can occur simultaneously. (+info)
Murine and human cathepsin Z: cDNA-cloning, characterization of the genes and chromosomal localization.
(11/284)
A novel murine cysteine protease from the family of papain-like cysteine proteinases was identified by dbEST-database search. A 1. 4-kb full-length cDNA encoding a predicted polypeptide of 306 amino acids was characterized. The new protease, tentatively named cathepsin Z, exhibits all features characteristics of a papain-like cysteine protease, including the highly conserved residues of the 'catalytic triad'. Cathepsin Z shares only 26-35% overall homology with previously described mammalian papain-like cysteine peptidases and has an unusually short propeptide, which may indicate that it is a member of a putative new subfamily within the family of papain-like cysteine peptidases. Genomic clones covering the murine and human cathepsin Z genes were isolated. They comprise six exons and five introns spanning a 12-kb region of genomic DNA, respectively. Murine cathepsin Z was mapped to chromosome 2, a region with synteny homology to a region of human chromosome 20 to which human cathepsin Z has been mapped previously. Northern blot analysis revealed ubiquitous expression of murine cathepsin Z. Multiple transcriptional start sites were identified for the murine cathepsin Z gene and together with the absence of a TATA box, a high G+C content, a CpG island and the presence of several Sp1-binding sites in the promoter region, murine cathepsin Z may be classified as a 'housekeeping' gene. (+info)
Mouse cathepsin M, a placenta-specific lysosomal cysteine protease related to cathepsins L and P.
(12/284)
The complete nucleotide sequence of a novel cathepsin cDNA derived from mouse placenta was determined and is termed cathepsin M. The predicted protein of 333 amino acid is a member of the family C1A proteases and is related to mouse cathepsins L and P. Mouse cathepsin M is highly expressed in placenta, whereas no detectable levels were found in lung, spleen, heart, brain, kidney, thymus, testicle, liver, or embryo. Phylogenic analyses of the sequences of human and mouse cathepsins show that cathepsin M is most closely related to cathepsins P and L. However, the differences are sufficiently large to indicate that the enzymes will be found in other species. This is in contrast to human cathepsins L and V, which probably resulted from a gene duplication after divergence of mammalian species. (+info)
Cathepsin K gene mutations and 1q21 haplotypes in at patients with pycnodysostosis in an outbred population.
(13/284)
The molecular genetics of the autosomal recessive disorder pycnodysostosis was studied in five independent families from an outbred Caucasian population. We found two new mutations and one recently described mutation in the cathepsin K gene by sequencing DNA from eight patients with pycnodysostosis: a one base transition in exon8, c926T > C, causing a single amino acid substitution leucine-->proline, L309P; A 3' splice site mutation in intron 2, c121-1G > A, causing deletion of all exon 3, 41V-81Mdel; and the exon 3 missense mutation c236G > A leading to residue G79E. In three of the families patients were homozygous for 926T > C. In the remaining two families patients were heterozygous for 926T > C and 121-1G > A in one case, and for 926T > C and 236G > A in the other case. Assays using genomic DNA were developed for all three mutations. We tested 150 healthy control persons and observed the mutation frequencies: 0 to 300 for 121-1G > A and 236G > A and 1 to 150 for 926T > C. One patient from each family was haplotyped with eight microsatellite markers surrounding the cathepsin K gene on chromosome 1q21. A very rare, P = 1.8 x 10(-6) to P = 0.0004, and highly preserved area around the presumed disease locus was common to all the patients. This haplotype was found on seven chromosomes identical by state, IBS, out of the possible eight carrying the 926T > C mutation. Founder effect, locus homogeneity, and allele heterogeneity regarding pycnodysostosis within this population are discussed. Finally, the first pregnancy and delivery described in a patient with pycnodysostosis is reported. (+info)
Fibroblast growth factor (FGF)-2 directly stimulates mature osteoclast function through activation of FGF receptor 1 and p42/p44 MAP kinase.
(14/284)
We previously reported that fibroblast growth factor-2 (FGF-2) acts not only on osteoblasts to stimulate osteoclastic bone resorption indirectly but also on mature osteoclasts directly. In this study, we investigated the mechanism of this direct action of FGF-2 on mature osteoclasts using mouse and rabbit osteoclast culture systems. FGF-2 stimulated pit formation resorbed by isolated rabbit osteoclasts moderately from low concentrations (>/=10(-12) m), whereas at high concentrations (>/=10(-9) m) it showed stimulation on pit formation resorbed by unfractionated bone cells very potently. FGF-2 (>/=10(-12) m) also increased cathepsin K and MMP-9 mRNA levels in mouse and rabbit osteoclasts. Among FGF receptors (FGFR1 to 4) only FGFR1 was detected on isolated mouse osteoclasts, whereas all FGFRs were identified on mouse osteoblasts. FGF-2 (>/=10(-12) m) up-regulated the phosphorylation of cellular proteins, including p42/p44 mitogen-activated protein (MAP) kinase, and increased the kinase activity of immunoprecipitated FGFR1 in mouse osteoclasts. The stimulation of FGF-2 on mouse and rabbit osteoclast functions was abrogated by PD-98059, a specific inhibitor of p42/p44 MAP kinase. These results strongly suggest that FGF-2 acts directly on mature osteoclasts through activation of FGFR1 and p42/p44 MAP kinase, causing the stimulation of bone resorption at physiological or pathological concentrations. (+info)
Cathepsin-6, a novel cysteine proteinase showing homology with and co-localized expression with cathepsin J/P in the labyrinthine layer of mouse placenta.
(15/284)
A novel cysteine proteinase, cathepsin-6, was isolated by RNA differential display from mouse placenta. Cathepsin-6 showed the highest homology with cathepsin J (same as P) and L. The structural features including the catalytic triad of the C1 proteinase family were well conserved in cathepsin-6. The expression of cathepsin-6 and cathepsin J/P was restricted in labyrinthine trophoblasts of the placenta. (+info)
Biochemical characterization of human cathepsin X revealed that the enzyme is an exopeptidase, acting as carboxymonopeptidase or carboxydipeptidase.
(16/284)
Cathepsin X, purified to homogeneity from human liver, is a single chain glycoprotein with a molecular mass of approximately 33 kDa and pI 5.1-5.3. Cathepsin X was inhibited by stefin A, cystatin C and chicken cystatin (Ki = 1.7-15.0 nM), but poorly or not at all by stefin B (Ki > 250 nM) and L-kininogen, respectively. The enzyme was also inhibited by two specific synthetic cathepsin B inhibitors, CA-074 and GFG-semicarbazone. Cathepsin X was similar to cathepsin B and found to be a carboxypeptidase with preference for a positively charged Arg in P1 position. Contrary to the preference of cathepsin B, cathepsin X normally acts as a carboxymonopeptidase. However, the preference for Arg in the P1 position is so strong that cathepsin X cleaves substrates with Arg in antepenultimate position, acting also as a carboxydipeptidase. A large hydrophobic residue such as Trp is preferred in the P1' position, although the enzyme cleaved all P1' residues investigated (Trp, Phe, Ala, Arg, Pro). Cathepsin X also cleaved substrates with amide-blocked C-terminal carboxyl group with rates similar to those of the unblocked substrates. In contrast, no endopeptidase activity of cathepsin X could be detected on a series of o-aminobenzoic acid-peptidyl-N-[2,-dinitrophenyl]ethylenediamine substrates. Furthermore, the standard cysteine protease methylcoumarine amide substrates (kcat/Km approximately 5.0 x 103 M-1.s-1) were degraded approximately 25-fold less efficiently than the carboxypeptidase substrates (kcat/Km approximately 120.0 x 103 M-1.s-1). (+info)