Silicatein α: Cathepsin L-like protein in sponge biosilica

Earth’s biota produces vast quantities of polymerized silica at ambient temperatures and pressures by mechanisms that are not understood. Silica spicules constitute 75% of the dry weight of the sponge Tethya aurantia, making this organism uniquely tractable for analyses of the proteins intimately associated with the biosilica. Each spicule contains a central protein filament, shown by x-ray diffraction to exhibit a highly regular, repeating structure. The protein filaments can be dissociated to yield three similar subunits, named silicatein α, β, and γ. The molecular weights and amino acid compositions of the three silicateins are similar, suggesting that they are members of a single protein family. The cDNA sequence of silicatein α, the most abundant of these subunits, reveals that this protein is highly similar to members of the cathepsin L and papain family of proteases. The cysteine at the active site in the proteases is replaced by serine in silicatein α, although the six cysteines that form disulfide bridges in the proteases are conserved. Silicatein α also contains unique tandem arrays of multiple hydroxyls. These structural features may help explain the mechanism of biosilicification and the recently discovered activity of the silicateins in promoting the condensation of silica and organically modified siloxane polymers (silicones) from the corresponding silicon alkoxides. They suggest the possibility of a dynamic role of the silicateins in silicification of the sponge spicule and offer the prospect of a new synthetic route to silica and siloxane polymers at low temperature and pressure and neutral pH.

Evidence from disruption of the lmcpb gene array of Leishmania mexicana that cysteine proteinases are virulence factors.

The mammalian form of the protozoan parasite Leishmania mexicana contains high activity of a cysteine proteinase (LmCPb) encoded on a tandem array of 19 genes (lmcpb). Homozygous null mutants for lmcpb have been produced by targeted gene disruption. All life-cycle stages of the mutant can be cultured in vitro, demonstrating that the gene is not essential for growth or differentiation of the parasite. However, the mutant exhibits a marked phenotype affecting virulence-- its infectivity to macrophages is reduced by 80%. The mutants are as efficient as wild-type parasites in invading macrophages but they only survive in a small proportion of the cells. However, those parasites that successfully infect these macrophages grow normally. Despite their reduced virulence, the mutants are still able to produce subcutaneous lesions in mice, albeit at a slower rate than wild-type parasites. The product of a single copy of lmcpb re-expressed in the null mutant was enzymatically active and restored infectivity toward macrophages to wild-type levels. Double null mutants created for lmcpb and lmcpa (another cathepsin L-like cysteine proteinase) have a similar phenotype to the lmcpb null mutant, showing that LmCPa does not compensate for the loss of LmCPb.

Retinoic acid alters the intracellular trafficking of the mannose-6-phosphate/insulin-like growth factor II receptor and lysosomal enzymes

Previously, we showed that retinoic acid (RA) binds to the mannose-6-phosphate/insulin-like growth factor II receptor (M6P/IGF2R) with high affinity, suggesting that M6P/IGF2R may be a receptor for RA. Here, we show that RA, after 2–3 h of incubation with cultured neonatal-rat cardiac fibroblasts, dramatically alters the intracellular distribution of M6P/IGF2R as well as that of cathepsin B (a lysosomal protease bearing M6P). Immunofluorescence techniques indicate that this change in intracellular distribution is characterized by a shift of the proteins from the perinuclear area to cytoplasmic vesicles. The effect of RA was neither blocked by an RA nuclear receptor antagonist (AGN193109) nor mimicked by a selective RA nuclear-receptor agonist (TTNPB). Furthermore, the RA-induced translocation of cathepsin B was not observed in M6P/IGF2R-deficient P388D1 cells but occurred in stably transfected P388D1 cells expressing the receptor, suggesting that the effect of RA might be the result of direct interaction with M6P/IGF2R, rather than the result of binding to the nuclear receptors. These observations not only support the idea that M6P/IGF2R mediates an RA-response pathway but also indicate a role for RA in control of intracellular trafficking of lysosomal enzymes. Therefore, our observations may have important implications for the understanding of the diverse biological effects of retinoids.

Loss of the gene encoding mannose 6-phosphate/insulin-like growth factor II receptor is an early event in liver carcinogenesis

This report shows that loss of heterozygosity at the mannose 6-phosphate/insulin-like growth factor II receptor (M6P/IGF2R) locus occurred in 5/8 (63%) dysplastic liver lesions and 11/18 (61%) hepatocellular carcinomas (HCCs) associated with the high risk factors of hepatitis virus infection and liver cirrhosis. Mutations in the remaining allele were detected in 6/11 (55%) HCCs, including deletions in a polydeoxyguanosine region known to be a target of microsatellite instability. M6P/IGF2R allele loss was also found in cirrhotic tissue of clonal origin adjacent to these dysplastic lesions and HCCs, demonstrating that M6P/IGF2R inactivation occurs early in liver carcinogenesis. In conclusion, HCCs frequently develop from clonal expansions of phenotypically normal, M6P/IGF2R-mutated hepatocytes, providing further support for the idea that M6P/IGF2R functions as a liver tumor-suppressor gene.

Chromosome-specific molecular organization of maize (Zea mays L.) centromeric regions

A set of oat–maize chromosome addition lines with individual maize (Zea mays L.) chromosomes present in plants with a complete oat (Avena sativa L.) chromosome complement provides a unique opportunity to analyze the organization of centromeric regions of each maize chromosome. A DNA sequence, MCS1a, described previously as a maize centromere-associated sequence, was used as a probe to isolate cosmid clones from a genomic library made of DNA purified from a maize chromosome 9 addition line. Analysis of six cosmid clones containing centromeric DNA segments revealed a complex organization. The MCS1a sequence was found to comprise a portion of the long terminal repeats of a retrotransposon-like repeated element, termed CentA. Two of the six cosmid clones contained regions composed of a newly identified family of tandem repeats, termed CentC. Copies of CentA and tandem arrays of CentC are interspersed with other repetitive elements, including the previously identified maize retroelements Huck and Prem2. Fluorescence in situ hybridization revealed that CentC and CentA elements are limited to the centromeric region of each maize chromosome. The retroelements Huck and Prem2 are dispersed along all maize chromosomes, although Huck elements are present in an increased concentration around centromeric regions. Significant variation in the size of the blocks of CentC and in the copy number of CentA elements, as well as restriction fragment length variations were detected within the centromeric region of each maize chromosome studied. The different proportions and arrangements of these elements and likely others provide each centromeric region with a unique overall structure.

Insulin-like growth factor I is a growth-promoting factor for Leishmania promastigotes and amastigotes

Leishmaniases are diseases caused by protozoa of the genus Leishmania that affect more than 20 million people in the world. The initial phase of the infection is fundamental for either the progression or control of the disease. The Leishmania parasites are injected in the skin as promastigotes and then, after been phagocytized by the host macrophages, rapidly transform into amastigotes. In this phase different nonspecific cellular and humoral elements participate. We have shown previously that insulin-like growth factor (IGF)-I that is constitutively present in the skin induces growth of Leishmania promastigotes. In the present paper we show further evidence for the importance of this factor: (i) IGF-I also can induce a growth response in Leishmania (Leishmania) mexicana amastigotes; (ii) IGF-I binds specifically to a putative single-site receptor on both promastigotes and amastigotes; (iii) IGF-I induces a rapid tyrosine phosphorylation of parasite proteins with different molecular mass in promastigotes and amastigotes of L. (L.) mexicana; and, finally, (iv) the cutaneous lesion in the mice when challenged by IGF-I-preactivated Leishmania (Viannia) panamensis is increased significantly because of inflammatory process and growth of parasites. We thus suggest that IGF-I is another important host factor participating in the Leishmania–host interplay in the early stage during the establishment of the infection and presumably also in the later stages.

Elevated free nitrotyrosine levels, but not protein-bound nitrotyrosine or hydroxyl radicals, throughout amyotrophic lateral sclerosis (ALS)-like disease implicate tyrosine nitration as an aberrant in vivo property of one familial ALS-linked superoxide dismutase 1 mutant

Mutations in superoxide dismutase 1 (SOD1; EC 1.15.1.1) are responsible for a proportion of familial amyotrophic lateral sclerosis (ALS) through acquisition of an as-yet-unidentified toxic property or properties. Two proposed possibilities are that toxicity may arise from imperfectly folded mutant SOD1 catalyzing the nitration of tyrosines [Beckman, J. S., Carson, M., Smith, C. D. & Koppenol, W. H. (1993) Nature (London) 364, 584] through use of peroxynitrite or from peroxidation arising from elevated production of hydroxyl radicals through use of hydrogen peroxide as a substrate [Wiedau-Pazos, M., Goto, J. J., Rabizadeh, S., Gralla, E. D., Roe, J. A., Valentine, J. S. & Bredesen, D. E. (1996) Science 271, 515–518]. To test these possibilities, levels of nitrotyrosine and markers for hydroxyl radical formation were measured in two lines of transgenic mice that develop progressive motor neuron disease from expressing human familial ALS-linked SOD1 mutation G37R. Relative to normal mice or mice expressing high levels of wild-type human SOD1, 3-nitrotyrosine levels were elevated by 2- to 3-fold in spinal cords coincident with the earliest pathological abnormalities and remained elevated in spinal cord throughout progression of disease. However, no increases in protein-bound nitrotyrosine were found during any stage of SOD1-mutant-mediated disease in mice or at end stage of sporadic or SOD1-mediated familial human ALS. When salicylate trapping of hydroxyl radicals and measurement of levels of malondialdehyde were used, there was no evidence throughout disease progression in mice for enhanced production of hydroxyl radicals or lipid peroxidation, respectively. The presence of elevated nitrotyrosine levels beginning at the earliest stages of cellular pathology and continuing throughout progression of disease demonstrates that tyrosine nitration is one in vivo aberrant property of this ALS-linked SOD1 mutant.

Arabidopsis thaliana PAD4 encodes a lipase-like gene that is important for salicylic acid signaling

The Arabidopsis PAD4 gene previously was found to be required for expression of multiple defense responses including camalexin synthesis and PR-1 gene expression in response to infection by the bacterial pathogen Pseudomonas syringae pv. maculicola. This report describes the isolation of PAD4. The predicted PAD4 protein sequence displays similarity to triacyl glycerol lipases and other esterases. The PAD4 transcript was found to accumulate after P. syringae infection or treatment with salicylic acid (SA). PAD4 transcript levels were very low in infected pad4 mutants. Treatment with SA induced expression of PAD4 mRNA in pad4–1, pad4–3, and pad4–4 plants but not in pad4–2 plants. Induction of PAD4 expression by P. syringae was independent of the regulatory factor NPR1 but induction by SA was NPR1-dependent. Taken together with the previous observation that pad4 mutants have a defect in accumulation of SA upon pathogen infection, these results suggest that PAD4 participates in a positive regulatory loop that increases SA levels, thereby activating SA-dependent defense responses.

Three members of a novel small gene-family from Arabidopsis thaliana able to complement functionally an Escherichia coli mutant defective in PAPS reductase activity encode proteins with a thioredoxin-like domain and “APS reductase” activity

Three different cDNAs, Prh-19, Prh-26, and Prh-43 [3′-phosphoadenosine-5′-phosphosulfate (PAPS) reductase homolog], have been isolated by complementation of an Escherichia coli cysH mutant, defective in PAPS reductase activity, to prototrophy with an Arabidopsis thaliana cDNA library in the expression vector λYES. Sequence analysis of the cDNAs revealed continuous open reading frames encoding polypeptides of 465, 458, and 453 amino acids, with calculated molecular masses of 51.3, 50.5, and 50.4 kDa, respectively, that have strong homology with fungal, yeast, and bacterial PAPS reductases. However, unlike microbial PAPS reductases, each PRH protein has an N-terminal extension, characteristic of a plastid transit peptide, and a C-terminal extension that has amino acid and deduced three-dimensional homology to thioredoxin proteins. Adenosine 5′-phosphosulfate (APS) was shown to be a much more efficient substrate than PAPS when the activity of the PRH proteins was tested by their ability to convert 35S-labeled substrate to acid-volatile 35S-sulfite. We speculate that the thioredoxin-like domain is involved in catalytic function, and that the PRH proteins may function as novel “APS reductase” enzymes. Southern hybridization analysis showed the presence of a small multigene family in the Arabidopsis genome. RNA blot hybridization with gene-specific probes revealed for each gene the presence of a transcript of ≈1.85 kb in leaves, stems, and roots that increased on sulfate starvation. To our knowledge, this is the first report of the cloning and characterization of plant genes that encode proteins with APS reductase activity and supports the suggestion that APS can be utilized directly, without activation to PAPS, as an intermediary substrate in reductive sulfate assimilation.

Stretched and overwound DNA forms a Pauling-like structure with exposed bases

We investigate structural transitions within a single stretched and supercoiled DNA molecule. With negative supercoiling, for a stretching force >0.3 pN, we observe the coexistence of B-DNA and denatured DNA from σ ≈ −0.015 down to σ = −1. Surprisingly, for positively supercoiled DNA (σ > +0.037) stretched by 3 pN, we observe a similar coexistence of B-DNA and a new, highly twisted structure. Experimental data and molecular modeling suggest that this structure has ≈2.62 bases per turn and an extension 75% larger than B-DNA. This structure has tightly interwound phosphate backbones and exposed bases in common with Pauling’s early DNA structure [Pauling, L. & Corey, R. B. (1953), Proc. Natl. Acad. Sci. USA 39, 84–97] and an unusual structure proposed for the Pf1 bacteriophage [Liu, D. J. & Day, L. A. (1994) Science 265, 671–674].

Tarsier-like locomotor specializations in the Oligocene primate Afrotarsius

Tarsiers and extinct tarsier-like primates have played a central role in views of primate phylogeny and evolution for more than a century. Because of the importance of tarsiers in so many primatological problems, there has been particular interest in questions about the origin of tarsier specializations and the biogeography of early tarsioid radiations. We report on a new fossil of rare Afrotarsius that shows near identity to modern Tarsius in unique specializations of the leg, which provides information about the locomotor behavior and clarifies the phylogenetic position of this previously controversial primate. These specializations constitute evidence that Afrotarsius is a tarsiid, closely related to extant Tarsius; hence, it is now excluded from being a generalized sister taxon to Anthropoidea.

An essential cell division gene of Drosophila, absent from Saccharomyces, encodes an unusual protein with tubulin-like and myosin-like peptide motifs

Null mutations at the misato locus of Drosophila melanogaster are associated with irregular chromosomal segregation at cell division. The consequences for morphogenesis are that mutant larvae are almost devoid of imaginal disk tissue, have a reduction in brain size, and die before the late third-instar larval stage. To analyze these findings, we isolated cDNAs in and around the misato locus, mapped the breakpoints of chromosomal deficiencies, determined which transcript corresponded to the misato gene, rescued the cell division defects in transgenic organisms, and sequenced the genomic DNA. Database searches revealed that misato codes for a novel protein, the N-terminal half of which contains a mixture of peptide motifs found in α-, β-, and γ-tubulins, as well as a motif related to part of the myosin heavy chain proteins. The sequence characteristics of misato indicate either that it arose from an ancestral tubulin-like gene, different parts of which underwent convergent evolution to resemble motifs in the conventional tubulins, or that it arose by the capture of motifs from different tubulin genes. The Saccharomyces cerevisiae genome lacks a true homolog of the misato gene, and this finding highlights the emerging problem of assigning functional attributes to orphan genes that occur only in some evolutionary lineages.

Rotavirus contains integrin ligand sequences and a disintegrin-like domain that are implicated in virus entry into cells

Rotavirus contains two outer capsid viral proteins, the spike protein VP4 and major capsid component VP7, both of which are implicated in cell entry. We show that VP4 and VP7 contain tripeptide sequences previously shown to act as recognition sites for integrins in extracellular matrix proteins. VP4 contains the α2β1 integrin ligand site DGE. In VP7, the αxβ2 integrin ligand site GPR and the α4β1 integrin ligand site LDV are embedded in a novel disintegrin-like domain that also shows sequence similarity to fibronectin and the tie receptor tyrosine kinase. Microorganism sequence homology to these ligand motifs and to disintegrins has not been reported previously. In our experiments, peptides including these rotaviral tripeptides and mAbs directed to these integrins specifically blocked rotavirus infection of cells shown to express α2β1 and β2 integrins. Rotavirus VP4-mediated cell entry may involve the α2β1 integrin, whereas VP7 appears to interact with αxβ2 and α4β1 integrins.

Impaired osteoclastic bone resorption leads to osteopetrosis in cathepsin-K-deficient mice

Cathepsin K is a recently identified lysosomal cysteine proteinase. It is abundant in osteoclasts, where it is believed to play a vital role in the resorption and remodeling of bone. Pycnodysostosis is a rare inherited osteochondrodysplasia that is caused by mutations of the cathepsin-K gene, characterized by osteosclerosis, short stature, and acroosteolysis of the distal phalanges. With a view to delineating the role of cathepsin K in bone resorption, we generated mice with a targeted disruption of this proteinase. Cathepsin-K-deficient mice survive and are fertile, but display an osteopetrotic phenotype with excessive trabeculation of the bone-marrow space. Cathepsin-K-deficient osteoclasts manifested a modified ultrastructural appearance: their resorptive surface was poorly defined with a broad demineralized matrix fringe containing undigested fine collagen fibrils; their ruffled borders lacked crystal-like inclusions, and they were devoid of collagen-fibril-containing cytoplasmic vacuoles. Assaying the resorptive activity of cathepsin-K-deficient osteoclasts in vitro revealed this function to be severely impaired, which supports the contention that cathepsin K is of major importance in bone remodeling.

Design of potent and selective human cathepsin K inhibitors that span the active site

Potent and selective active-site-spanning inhibitors have been designed for cathepsin K, a cysteine protease unique to osteoclasts. They act by mechanisms that involve tight binding intermediates, potentially on a hydrolytic pathway. X-ray crystallographic, MS, NMR spectroscopic, and kinetic studies of the mechanisms of inhibition indicate that different intermediates or transition states are being represented that are dependent on the conditions of measurement and the specific groups flanking the carbonyl in the inhibitor. The species observed crystallographically are most consistent with tetrahedral intermediates that may be close approximations of those that occur during substrate hydrolysis. Initial kinetic studies suggest the possibility of irreversible and reversible active-site modification. Representative inhibitors have demonstrated antiresorptive activity both in vitro and in vivo and therefore are promising leads for therapeutic agents for the treatment of osteoporosis. Expansion of these inhibitor concepts can be envisioned for the many other cysteine proteases implicated for therapeutic intervention.