Autosomal-Recessive Posterior Microphthalmos Is Caused by Mutations in PRSS56, a Gene Encoding a Trypsin-Like Serine Protease.

Posterior microphthalmos (MCOP) is a rare isolated developmental anomaly of the eye characterized by extreme hyperopia due to short axial length. The population of the Faroe Islands shows a high prevalence of an autosomal-recessive form (arMCOP) of the disease. Based on published linkage data, we refined the position of the disease locus (MCOP6) in an interval of 250 kb in chromosome 2q37.1 in two large Faroese families. We detected three different mutations in PRSS56. Patients of the Faroese families were either homozygous for c.926G>C (p.Trp309Ser) or compound heterozygous for c.926G>C and c.526C>G (p.Arg176Gly), whereas a homozygous 1 bp duplication (c.1066dupC) was identified in five patients with arMCOP from a consanguineous Tunisian family. In one patient with MCOP from the Faroe Islands and in another one from Turkey, no PRSS56 mutation was detected, suggesting nonallelic heterogeneity of the trait. Using RT-PCR, PRSS56 transcripts were detected in samples derived from the human adult retina, cornea, sclera, and optic nerve. The expression of the mouse ortholog could be first detected in the eye at E17 and was maintained into adulthood. The predicted PRSS56 protein is a 603 amino acid long secreted trypsin-like serine peptidase. The c.1066dupC is likely to result in a functional null allele, whereas the two point mutations predict the replacement of evolutionary conserved and functionally important residues. Molecular modeling of the p.Trp309Ser mutant suggests that both the affinity and reactivity of the enzyme toward in vivo protein substrates are likely to be substantially reduced.

Comparison of purified 12 kDa and recombinant 15 kDa Fasciola hepatica antigens related to a Schistosoma mansoni fatty acid binding protein

Vaccines in schistosomiasis using homologous antigens have been studied extensively in experimentally infected mammalian hosts. Vaccines using heterologous antigens have received comparatively less attention. This review summarizes recent work on a heterologous 12 kDa Fasciola hepatica antigenic polypeptide which cross reacts with Schistosoma mansoni. A cDNA has been cloned and sequenced, and the predicted amino acid sequence of the recombinant protein has been shown to have significant (44) identity with a 14 kDa S. mansoni fatty acid binding protein. Thus in the parasitic trematodes fatty acid binding proteins may be potential vaccine candidates. The F. hepatica recombinant protein has been overexpressed and purified and denoted rFh15. Preliminary rFh15 migrates more slowly (i.e. may be slightly larger) than nFh12 on SDS-PAGE and has a predicted pI of 6.01 vs. observed pI of 5.45. Mice infected with F. hepatica develop antibodies to nFh12 by 2 weeks of infection vs. 6 weeks of infection to rFh15; on the other hand, mice with schistosomiasis mansoni develop antibodies to both nFh12 and rFh15 by 6 weeks of infection. Both the F. hepatica and S. mansoni cross-reactive antigens may be cross-protective antigens with the protection inducing capability against both species.

The influence of sugars and amino acids on the blood-feeding behaviour, oviposition and longevity of laboratory colony of Lutzomyia longipalpis (Lutz & Neiva, 1912) (Diptera: Psychodidae, Phlebotominae)

Schneider's Drosophila medium, a complex amino acid rich medium was tested alone and with seven different sugars for some aspects of the biology of Lutzomyia longipalpis. Statistically significant results were obtained when sucrose was used alone, indicating that among the sugars tested, this is still the most suitable and practical one for the maintenance of L. longipalpis colonies. However, the addition of Schneider's medium to a pool of different sugars, was suggested to be related with the acceptance of the first and second blood meals and to longevity, these being, obviously, quite relevant aspects when tansmission experiments are contemplated.

Progesterone down-regulates the open probability of the amiloride-sensitive epithelial sodium channel via a Nedd4-2-dependent mechanism.

Activation of the mitogen-activated protein (MAP) kinase cascade by progesterone in Xenopus oocytes leads to a marked down-regulation of activity of the amiloride-sensitive epithelial sodium channel (ENaC). Here we have studied the signaling pathways involved in progesterone effect on ENaC activity. We demonstrate that: (i) the truncation of the C termini of the alphabetagammaENaC subunits results in the loss of the progesterone effect on ENaC; (ii) the effect of progesterone was also suppressed by mutating conserved tyrosine residues in the Pro-X-X-Tyr (PY) motif of the C termini of the beta and gamma ENaC subunits (beta(Y618A) and gamma(Y628A)); (iii) the down-regulation of ENaC activity by progesterone was also suppressed by co-expression ENaC subunits with a catalytically inactive mutant of Nedd4-2, a ubiquitin ligase that has been previously demonstrated to decrease ENaC cell-surface expression via a ubiquitin-dependent internalization/degradation mechanism; (iv) the effect of progesterone was significantly reduced by suppression of consensus sites (beta(T613A) and gamma(T623A)) for ENaC phosphorylation by the extracellular-regulated kinase (ERK), a MAP kinase previously shown to facilitate the binding of Nedd4 ubiquitin ligases to ENaC; (v) the quantification of cell-surface-expressed ENaC subunits revealed that progesterone decreases ENaC open probability (whole cell P(o), wcP(o)) and not its cell-surface expression. Collectively, these results demonstrate that the binding of active Nedd4-2 to ENaC is a crucial step in the mechanism of ENaC inhibition by progesterone. Upon activation of ERK, the effect of Nedd4-2 on ENaC open probability can become more important than its effect on ENaC cell-surface expression.

Dihydroaeruginoic acid synthetase and pyochelin synthetase, products of the pchEF genes, are induced by extracellular pyochelin in Pseudomonas aeruginosa.

The siderophore pyochelin of Pseudomonas aeruginosa is derived from one molecule of salicylate and two molecules of cysteine. Two cotranscribed genes, pchEF, encoding peptide synthetases have been identified and characterized. pchE was required for the conversion of salicylate to dihydroaeruginoate (Dha), the condensation product of salicylate and one cysteine residue and pchF was essential for the synthesis of pyochelin from Dha. The deduced PchE (156 kDa) and PchF (197 kDa) proteins had adenylation, thiolation and condensation/cyclization motifs arranged as modules which are typical of those peptide synthetases forming thiazoline rings. The pchEF genes were coregulated with the pchDCBA operon, which provides enzymes for the synthesis (PchBA) and activation (PchD) of salicylate as well as a putative thioesterase (PchC). Expression of a translational pchE'-'lacZ fusion was strictly dependent on the PchR regulator and was induced by extracellular pyochelin, the end product of the pathway. Iron replete conditions led to Fur (ferric uptake regulator)-dependent repression of the pchE'-'lacZ fusion. A translational pchD'-'lacZ fusion was also positively regulated by PchR and pyochelin and repressed by Fur and iron. Thus, autoinduction by pyochelin (or ferric pyochelin) and repression by iron ensure a sensitive control of the pyochelin pathway in P. aeruginosa.

Limb-girdle Muscular Dystrophy Type 2A Can Result from Accelerated Autoproteolytic Inactivation of Calpain 3.

Loss-of-function mutations in calpain 3 have been shown to cause limb-girdle muscular dystrophy type 2A (LGMD2A), an autosomal recessive disorder that results in gradual wasting of the muscles of the hip and shoulder areas. Due to the inherent instability of calpain 3, recombinant expression of the full-length enzyme has not been possible, making in vitro analysis of specific LGMD2A-causing mutations difficult. However, because calpain 3 is highly similar in amino acid sequence to calpain 2, the recently solved crystal structure of full-length, Ca2+-bound, calpastatin-inhibited rat calpain 2 has allowed us to model calpain 3 as a Ca2+-bound homodimer. The model revealed three distinct areas of the enzyme that undergo a large conformational change upon Ca2+-binding. Located in these areas are several residues that undergo mutation to cause LGMD2A. We investigated the in vitro effects of six of these mutations by making the corresponding mutations in rat calpain 2. All six mutations examined in this study resulted in a decrease in enzyme activity. All but one of the mutations caused an increased rate of autoproteolytic degradation of the enzyme as witnessed by SDS-PAGE, indicating the decrease in enzyme activity is caused, at least in part, by an increase in the rate of autoproteolytic degradation. The putative in vivo effects of these mutations on calpain 3 activity are discussed with respect to their ability to cause LGMD2A.

Functional analysis and structural modeling of human APOBEC3G reveal the role of evolutionarily conserved elements in the inhibition of human immunodeficiency virus type 1 infection and Alu transposition.

Retroelements are important evolutionary forces but can be deleterious if left uncontrolled. Members of the human APOBEC3 family of cytidine deaminases can inhibit a wide range of endogenous, as well as exogenous, retroelements. These enzymes are structurally organized in one or two domains comprising a zinc-coordinating motif. APOBEC3G contains two such domains, only the C terminal of which is endowed with editing activity, while its N-terminal counterpart binds RNA, promotes homo-oligomerization, and is necessary for packaging into human immunodeficiency virus type 1 (HIV-1) virions. Here, we performed a large-scale mutagenesis-based analysis of the APOBEC3G N terminus, testing mutants for (i) inhibition of vif-defective HIV-1 infection and Alu retrotransposition, (ii) RNA binding, and (iii) oligomerization. Furthermore, in the absence of structural information on this domain, we used homology modeling to examine the positions of functionally important residues and of residues found to be under positive selection by phylogenetic analyses of primate APOBEC3G genes. Our results reveal the importance of a predicted RNA binding dimerization interface both for packaging into HIV-1 virions and inhibition of both HIV-1 infection and Alu transposition. We further found that the HIV-1-blocking activity of APOBEC3G N-terminal mutants defective for packaging can be almost entirely rescued if their virion incorporation is forced by fusion with Vpr, indicating that the corresponding region of APOBEC3G plays little role in other aspects of its action against this pathogen. Interestingly, residues forming the APOBEC3G dimer interface are highly conserved, contrasting with the rapid evolution of two neighboring surface-exposed amino acid patches, one targeted by the Vif protein of primate lentiviruses and the other of yet-undefined function.

Diethyldithiocarbamic acid inhibits the octadecanoid signaling pathway for the wound induction of proteinase-inhibitors in tomato leaves

The induction of proteinase inhibitor I synthesis in tomato (Lycopersicon esculentum) leaves in response to wounding is strongly inhibited by diethyldithiocarbamic acid (DIECA). DIECA also inhibits the induction of inhibitor I synthesis by the 18-amino acid polypeptide systemin, polygalacturonic acid (PCA), and linolenic acid, but not by jasmonic acid, suggesting that DIECA interferes with the octadecanoid signaling pathway. DIECA only weakly inhibited tomato lipoxygenase activity, indicating that DIECA action occurred at a step after the conversion of linolenic acid to 13(S)-hydroperoxylinolenic acid (HPOTrE). DIECA was shown to efficiently reduce HPOTrE to 13-hydroxylinolenic acid (HOTrE), which is not a signaling intermediate. Therefore, in vivo, DIECA is likely inhibiting the signaling pathway by shunting HPOTrE to HOTrE, thereby severely reducing the precursor pool leading to cyclization and eventual synthesis of jasmonic acid. Phenidone, an inhibitor of lipoxygenase, inhibited proteinase inhibitor I accumulation in response to wounding, further supporting a role for its substrate, linolenic acid, and its product, HPOTrE, as components of the signal-transduction pathway that induces proteinase inhibitor synthesis in response to wounding, systemin, and PCA.

Differential roles of T cell receptor alpha and beta chains in ligand binding among H-2Kd-restricted cytolytic T lymphocyte clones specific for a photoreactive Plasmodium berghei circumsporozoite peptide derivative.

To study the interaction of T cell receptor with its ligand, a complex of a major histocompatibility complex molecule and a peptide, we derived H-2Kd-restricted cytolytic T lymphocyte clones from mice immunized with a Plasmodium berghei circumsporozoite peptide (PbCS) 252-260 (SYIPSAEKI) derivative containing photoreactive Nepsilon-[4-azidobenzoyl] lysine in place of Pro-255. This residue and Lys-259 were essential parts of the epitope recognized by these clones. Most of the clones expressed BV1S1A1 encoded beta chains along with specific complementary determining region (CDR) 3beta regions but diverse alpha chain sequences. Surprisingly, all T cell receptors were preferentially photoaffinity labeled on the alpha chain. For a representative T cell receptor, the photoaffinity labeled site was located in the Valpha C-strand. Computer modeling suggested the presence of a hydrophobic pocket, which is formed by parts of the Valpha/Jalpha C-, F-, and G-strands and adjacent CDR3alpha residues and structured to be able to avidly bind the photoreactive ligand side chain. We previously found that a T cell receptor specific for a PbCS peptide derivative containing this photoreactive side chain in position 259 similarly used a hydrophobic pocket located between the junctional CDR3 loops. We propose that this nonpolar domain in these locations allow T cell receptors to avidly and specifically bind epitopes containing non-peptidic side chains.

A novel role for proline- and acid-rich basic region leucine zipper (PAR bZIP) proteins in the transcriptional regulation of a BH3-only proapoptotic gene.

Proline- and acid-rich (PAR) basic region leucine zipper (bZIP) proteins thyrotroph embryonic factor (TEF), D-site-binding protein (DBP), and hepatic leukemia factor have been involved in neurotransmitter homeostasis and amino acid metabolism. Here we demonstrate a novel role for these proteins in the transcriptional control of a BH3-only gene. PAR bZIP proteins are able to transactivate the promoter of bcl-gS. This promoter is particularly responsive to TEF activation and is silenced by NFIL3, a repressor that shares the consensus binding site with PAR bZIP proteins. Consistently, transfection of TEF induces the expression of endogenous bcl-gS in cancer cells, and this induction is independent of p53. A naturally occurring variant of DBP (tDBP), lacking the transactivation domain, has been identified and shown to impede the formation of active TEF dimers in a competitive manner and to reduce the TEF-dependent induction of bcl-gS. Of note, treatment of cancer cells with etoposide induces TEF activation and promotes the expression of bcl-gS. Furthermore, blockade of bcl-gS or TEF expression by a small interfering RNA strategy or transfection with tDBP significantly reduces the etoposide-mediated apoptotic cell death. These findings represent the first described role for PAR bZIP proteins in the regulation of a gene involved in the execution of apoptosis.

An mRNA region of the canine distemper virus fusion protein gene lacking AUG codons can promote protein expression.

Canine distemper virus (CDV) produces a glycosylated type I fusion protein (F) with an internal hydrophobic signal sequence beginning around 115 residues downstream of the first AUG used for translation initiation. Cleavage of the signal sequence yields the F0 molecule, which is cleaved into the F1 and F2 subunits. Surprisingly, when all in-frame AUGs located in the first third of the F gene were mutated a protein of the same molecular size as the F0 molecule was still expressed from both the Onderstepoort (OP) and A75/17-CDV F genes. We designated this protein, which is initiated from a non-AUG codon protein Fx. Site-directed mutagenesis allowed to identify codon 85, a GCC codon coding for alanine, as the most likely position from which translation initiation of Fx occurs in OP-CDV. Deletion analysis demonstrated that at least 60 nucleotides upstream of the GCC codon are required for efficient Fx translation. This sequence is GC-rich, suggesting extensive folding. Secondary structure may therefore be important for translation initiation at codon 85.

Characterization of a Bacillus subtilis thermosensitive teichoic acid-deficient mutant: gene mnaA (yvyH) encodes the UDP-N-acetylglucosamine 2-epimerase.

The Bacillus subtilis thermosensitive mutant ts-21 bears two C-G-->T-A transitions in the mnaA gene. At the nonpermissive temperature it is characterized by coccoid cell morphology and reduced cell wall phosphate content. MnaA converts UDP-N-acetylglucosamine into UDP-N-acetylmannosamine, a precursor of the teichoic acid linkage unit.

Enzymic, phylogenetic, and structural characterization of the unusual papain-like protease domain of Plasmodium falciparum SERA5.

Serine repeat antigen 5 (SERA5) is an abundant antigen of the human malaria parasite Plasmodium falciparum and is the most strongly expressed member of the nine-gene SERA family. It appears to be essential for the maintenance of the erythrocytic cycle, unlike a number of other members of this family, and has been implicated in parasite egress and/or erythrocyte invasion. All SERA proteins possess a central domain that has homology to papain except in the case of SERA5 (and some other SERAs), where the active site cysteine has been replaced with a serine. To investigate if this domain retains catalytic activity, we expressed, purified, and refolded a recombinant form of the SERA5 enzyme domain. This protein possessed chymotrypsin-like proteolytic activity as it processed substrates downstream of aromatic residues, and its activity was reversed by the serine protease inhibitor 3,4-diisocoumarin. Although all Plasmodium SERA enzyme domain sequences share considerable homology, phylogenetic studies revealed two distinct clusters across the genus, separated according to whether they possess an active site serine or cysteine. All Plasmodia appear to have at least one member of each group. Consistent with separate biological roles for members of these two clusters, molecular modeling studies revealed that SERA5 and SERA6 enzyme domains have dramatically different surface properties, although both have a characteristic papain-like fold, catalytic cleft, and an appropriately positioned catalytic triad. This study provides impetus for the examination of SERA5 as a target for antimalarial drug design.

Five new CYLD mutations in skin appendage tumors and evidence that aspartic acid 681 in CYLD is essential for deubiquitinase activity.

Brooke-Spiegler syndrome, familial cylindromatosis, and familial trichoepithelioma are autosomal-dominant genetic predispositions for benign tumors of skin appendages caused by mutations in the CYLD gene localized on chromosome 16q12-q13. The encoded protein functions as ubiquitin-specific protease (UBP), which negatively regulates NF-kappaB and c-Jun N-terminal kinase (JNK) signaling. We investigated five families affected with these skin neoplasms and identified four premature stop codons and the novel missense mutation D681G in a family in which 11 of 12 investigated tumors were trichoepitheliomas. CYLD protein harboring this missense mutation had a significant reduced ability to inhibit TNF receptor-associated factor (TRAF)2- and TRAF6-mediated NF-kappaB activation, tumor necrosis factor-alpha (TNFalpha)-induced JNK signaling, and to deubiquitinate TRAF2. CYLD-D681G was coimmunoprecipitated by TRAF2, but was unable to cleave K63-linked polyubiquitin chains. Aspartic acid 681 is highly conserved in CYLD homologues and other members of the UBP family, but does not belong to the Cys and His boxes providing the CYLD catalytic triad (Cys601, His871, and Asp889). As reported previously, the homologous residue D295 of HAUSP/USP-7 forms a hydrogen bond with the C-terminal end of ubiquitin and is important for the enzymatic activity. These results underline that D681 in CYLD is required for cleavage of K63-linked polyubiquitin chains.

Specific mutations in the estrogen receptor change the properties of antiestrogens to full agonists.

The estrogen receptor (ER) stimulates transcription of target genes by means of its two transcriptional activation domains, AF-1 in the N-terminal part of the receptor and AF-2 in its ligand-binding domain. AF-2 activity is dependent upon a putative amphipathic alpha-helix between residues 538 and 552 in the mouse ER. Point mutagenesis of conserved hydrophobic residues within this region reduces estrogen-dependent transcriptional activation without affecting hormone and DNA binding significantly. Here we show that these mutations dramatically alter the pharmacology of estrogen antagonists. Both tamoxifen and ICI 164,384 behave as strong agonists in HeLa cells expressing the ER mutants. In contrast to the wild-type ER, the mutant receptors maintain nuclear localization and DNA-binding activity after ICI 164,384 treatment. Structural alterations in AF-2 caused by gene mutations such as those described herein or by estrogen-independent signaling pathways may account for the insensitivity of some breast cancers to tamoxifen treatment.