A molecular model of the full-length human NOD-like receptor family CARD domain containing 5 (NLRC5) protein.

BACKGROUND: Pattern recognition receptors of the immune system have key roles in the regulation of pathways after the recognition of microbial- and danger-associated molecular patterns in vertebrates. Members of NOD-like receptor (NLR) family typically function intracellularly. The NOD-like receptor family CARD domain containing 5 (NLRC5) is the largest member of this family that also contains the largest number of leucine-rich repeats (LRRs).Due to the lack of crystal structures of full-length NLRs, projects have been initiated with the aim to model certain or all members of the family, but systematic studies did not model the full-length NLRC5 due to its unique domain architecture.Our aim was to analyze the LRR sequences of NLRC5 and some NLRC5-related proteins and to build a model for the full-length human NLRC5 by homology modeling. RESULTS: LRR sequences of NLRC5 were aligned and were compared with the consensus pattern of ribonuclease inhibitor protein (RI)-like LRR subfamily. Two types of alternating consensus patterns previously identified for RI repeats were also found in NLRC5. A homology model for full-length human NLRC5 was prepared and, besides the closed conformation of monomeric NLRC5, a heptameric platform was also modeled for the opened conformational NLRC5 monomers. CONCLUSIONS: Identification of consensus patterns of leucine-rich repeat sequences helped to identify LRRs in NLRC5 and to predict their number and position within the protein. In spite of the lack of fully adequate template structures, the presence of an untypical CARD domain and unusually high number of LRRs in NLRC5, we were able to construct a homology model for both the monomeric and homo-heptameric full-length human NLRC5 protein.

Protein phosphatase CaPpz1 is involved in cation homeostasis, cell wall integrity and virulence of Candida albicans.

The opportunistic pathogen Candida albicans has a single protein phosphatase Z (PPZ) candidate gene termed CaPPZ1, which shows significant allele variability. We demonstrate here that bacterially expressed CaPpz1 protein exhibits phosphatase activity which can be inhibited by recombinant Hal3, a known inhibitor of Saccharomyces cerevisiae Ppz1. Site-directed mutagenesis experiments based on natural polymorphisms allowed the identification of three amino acid residues that affect enzyme activity or stability. The expression of CaPPZ1 in ppz1 S. cerevisiae and pzh1 Schizosaccharomyces pombe cells partially rescued the salt and caffeine phenotypes of the deletion mutants. CaPpz1 also complemented the slt2 S. cerevisiae mutant, which is crippled in the mitogen-activated protein (MAP) kinase that mediates the cell wall integrity signalling pathway. Collectively, our results suggest that the orthologous PPZ enzymes have similar but not identical functions in different fungi. The deletion of the CaPPZ1 gene in C. albicans resulted in a mutant that was sensitive to salts such as LiCl and KCl, to caffeine, and to agents that affect cell wall biogenesis such as Calcofluor White and Congo red, but was tolerant to spermine and hygromycin B. Reintegration of the CaPPZ1 gene into the deletion mutant alleviated all of the mutant phenotypes tested. Thus CaPpz1 is involved in cation homeostasis, cell wall integrity and the regulation of the membrane potential of C. albicans. In addition, the germ tube growth rate, and virulence in the BALB/c mouse model, were reduced in the null mutant, suggesting a novel function for CaPpz1 in the yeast to hypha transition that may have medical relevance.

Molecular genetic characterization of novel sphingomyelin phosphodiesterase 1 mutations causing niemann-pick disease.

Niemann-Pick disease (NPD) types A and B are autosomal recessive disorders caused by acid sphingomyelinase (ASM) deficiency due to mutation in the sphingomyelin phosphodiesterase 1 gene (SMPD1). Although a number of SMPD1 mutations were reported, expression studies were performed for only a small number of missense mutations. We evaluated three unrelated patients with clinical manifestations of NPD. Sequence analysis revealed two previously described (S248R and W391G) and two novel (G247D and F572L) missense mutations. To analyze the effects of the novel mutations on ASM function, cDNA was generated by site-directed mutagenesis and expressed in COS-7 cells. In vitro biochemical assays revealed marked deficiency of ASM activity consistent with the disease phenotype in cells homoallelic for each mutation. We show that each mutation dramatically reduced half-life and catalytic activity of ASM with more pronounced decrease by the G247D mutation. These data suggest that impaired protein stability and decreased enzyme activity are responsible for the disease in sphingomyelinase-deficient patients carrying the G247D and F572L mutations.

A single conformational transglutaminase 2 epitope contributed by three domains is critical for celiac antibody binding and effects.

The multifunctional, protein cross-linking transglutaminase 2 (TG2) is the main autoantigen in celiac disease, an autoimmune disorder with defined etiology. Glutamine-rich gliadin peptides from ingested cereals, after their deamidation by TG2, induce T-lymphocyte activation accompanied by autoantibody production against TG2 in 1-2% of the population. The pathogenic role and exact binding properties of these antibodies to TG2 are still unclear. Here we show that antibodies from different celiac patients target the same conformational TG2 epitope formed by spatially close amino acids of adjacent domains. Glu153 and 154 on the first alpha-helix of the core domain and Arg19 on first alpha-helix of the N-terminal domain determine the celiac epitope that is accessible both in the closed and open conformation of TG2 and dependent on the relative position of these helices. Met659 on the C-terminal domain also can cooperate in antibody binding. This composite epitope is disease-specific, recognized by antibodies derived from celiac tissues and associated with biological effects when passively transferred from celiac mothers into their newborns. These findings suggest that celiac antibodies are produced in a surface-specific way for which certain homology of the central glutamic acid residues of the TG2 epitope with deamidated gliadin peptides could be a structural basis. Monoclonal mouse antibodies with partially overlapping epitope specificity released celiac antibodies from patient tissues and antagonized their harmful effects in cell culture experiments. Such antibodies or similar specific competitors will be useful in further functional studies and in exploring whether interference with celiac antibody actions leads to therapeutic benefits.

Computer-aided subsite mapping of α-amylases.

Subsite mapping is a crucial procedure in the characterization of α-amylases (EC 3.2.1.1), which are extensively used in starch-based industries and in diagnosis of pancreatic and salivary glands disorders. A computer-aided method has been developed for subsite mapping of α-amylases, which substitutes the difficult, expensive, and time-consuming experimental determination of action patterns to crystal structures based energy calculations. Interaction energies between enzymes and carbohydrate substrates were calculated after short energy minimization by a molecular mechanics program. A training set of wild type and mutant amylases with known experimental action patterns of 13 enzymes of wide range of origin was used to set up the procedure. Calculations for training set resulted in good correlation in case of subsite binding energies (r(2)=0.827-0.929) and bond cleavage frequencies (r(2)=0.727-0.835). A set of eight novel barley amylase 1 mutants was used to test our model. Subsite binding energies were predicted with r(2)=0.502 correlation coefficient, while bond cleavage frequency prediction resulted in r(2)=0.538. Our computer-aided procedure may supplement the experimental subsite mapping methods to predict and understand characteristic features of α-amylases.

The substrate specificity of Metarhizium anisopliae and Bos taurus carboxypeptidases A: insights into their use as tools for the removal of affinity tags.

Carboxypeptidases may serve as tools for removal of C-terminal affinity tags. In the present study, we describe the expression and purification of an A-type carboxypeptidase from the fungal pathogen Metarhizium anisopliae (MeCPA) that has been genetically engineered to facilitate the removal of polyhistidine tags from the C-termini of recombinant proteins. A complete, systematic analysis of the specificity of MeCPA in comparison with that of bovine carboxypeptidase A (BoCPA) was carried out. Our results indicate that the specificity of the two enzymes is similar but not identical. Histidine residues are removed more efficiently by MeCPA. The very inefficient digestion of peptides with C-terminal lysine or arginine residues, along with the complete inability of the enzyme to remove a C-terminal proline, suggests a strategy for designing C-terminal affinity tags that can be trimmed by MeCPA (or BoCPA) to produce a digestion product with a homogeneous endpoint.

Discovery and significance of new human T-lymphotropic viruses: HTLV-3 and HTLV-4.

Human T-lymphotropic virus type 1 (HTLV-1) and type 2 (HTLV-2) were discovered approximately 30 years ago and they are associated with various lymphoproliferative and neurological diseases. The estimated number of infected people is 10-20 million worldwide. In 2005, two new HTLV-1/HTLV-2-related viruses were detected, HTLV-3 and HTLV-4, from the same geographical area of Africa. In the last 4 years, their complete genomic sequences were determined and some of their characteristic features were studied in detail. These newly discovered retroviruses alongside their human (HTLV-1 and -2) and animal relatives (simian T-lymphotropic virus type 1-3) are reviewed. The potential risks associated with these viruses and the potential antiretroviral therapies are also discussed.

Drug targets in human T-lymphotropic virus type 1 (HTLV-1) infection.

Human T-lymphotropic virus type 1 (HTLV-1), the first known human retrovirus, induces various human diseases with a long latency period. The mechanism by which the virus causes diseases is still unknown. Studies indicate that viral replication is important at least for the development of HTLV-1 associated myelopathy, and therefore treatments based on our knowledge of human immunodeficiency virus type-1 (HIV-1) can be utilized to develop potent antiretroviral therapies targeting the replication enzymes reverse transcriptase, protease and integrase as well as the envelope glycoproteins. Furthermore, accessory gene products such as Tax and HBZ may also provide targets for chemotherapy. Treatment targeting these viral proteins may prevent the development of other HTLV-1-related diseases including adult T-cell leukemia, although such treatment may not be useful during the progression of the disease. This review describes the characteristics of HTLV-1 replication enzymes, envelope glycoproteins, and accessory proteins Tax and HBZ, and discusses the status of drug development strategies.

Substrate preference of transglutaminase 2 revealed by logistic regression analysis and intrinsic disorder examination.

Tissue transglutaminase (TG2) catalyzes the Ca(2+)-dependent posttranslational modification of proteins via formation of isopeptide bonds between their glutamine and lysine residues. Although substrate specificity of TG2 has been studied repeatedly at the sequence level, no clear consensus sequences have been determined so far. With the use of the extensive structural information on TG2 substrate proteins listed in TRANSDAB Wiki database, a slight preference of TG2 for glutamine and lysine residues situated in turns could be observed. When the spatial environment of the favored glutamine and lysine residues was analyzed with logistic regression, the presence of specific amino acid patterns was identified. By using the occurrence of the predictor amino acids as selection criteria, several polypeptides were predicted and later identified as novel in vitro substrates for TG2. By studying the sequence of TG2 substrate proteins lacking available crystal structure, the strong favorable influence on substrate selection of the presence of substrate glutamine and lysine residues in intrinsically disordered regions could also be revealed. The collected structural data have provided novel understanding of how this versatile enzyme selects its substrates in various cell compartments and tissues.

Amino acid preferences of retroviral proteases for amino-terminal positions in a type 1 cleavage site.

The specificities of the proteases of 11 retroviruses were studied using a series of oligopeptides with amino acid substitutions in the P1, P3, and P4 positions of a naturally occurring type 1 cleavage site (Val-Ser-Gln-Asn-Tyr downward arrowPro-Ile-Val-Gln) in human immunodeficiency virus type 1 (HIV-1). Previously, the substrate specificity of the P2 site was studied for the same representative set of retroviral proteases, which included at least one member from each of the seven genera of the family Retroviridae (P. Bagossi, T. Sperka, A. Fehér, J. Kádas, G. Zahuczky, G. Miklóssy, P. Boross, and J. Tözsér, J. Virol. 79:4213-4218, 2005). Our enzyme set comprised the proteases of HIV-1, HIV-2, equine infectious anemia virus, avian myeloblastosis virus (AMV), Mason-Pfizer monkey virus, mouse mammary tumor virus (MMTV), Moloney murine leukemia virus, human T-lymphotropic virus type 1, bovine leukemia virus, walleye dermal sarcoma virus, and human foamy virus. Molecular models were used to interpret the similarities and differences in specificity between these retroviral proteases. The results showed that the retroviral proteases had similar preferences (Phe and Tyr) for the P1 position in this sequence context, but differences were found for the P3 and P4 positions. Importantly, the sizes of the P3 and P4 residues appear to be a major contributor for specificity. The substrate specificities correlated well with the phylogenetic tree of the retroviruses. Furthermore, while the specificities of some enzymes belonging to different genera appeared to be very similar (e.g., those of AMV and MMTV), the specificities of the primate lentiviral proteases substantially differed from that observed for a nonprimate lentiviral protease.

C-terminal residues of mature human T-lymphotropic virus type 1 protease are critical for dimerization and catalytic activity.

HTLV-1 [HTLV (human T-cell lymphotrophic virus) type 1] is associated with a number of human diseases. HTLV-1 protease is essential for virus replication, and similarly to HIV-1 protease, it is a potential target for chemotherapy. The primary sequence of HTLV-1 protease is substantially longer compared with that of HIV-1 protease, and the role of the ten C-terminal residues is controversial. We have expressed C-terminally-truncated forms of HTLV-1 protease with and without N-terminal His tags. Removal of five of the C-terminal residues caused a 4-40-fold decrease in specificity constants, whereas the removal of an additional five C-terminal residues rendered the protease completely inactive. The addition of the N-terminal His tag dramatically decreased the activity of HTLV-1 protease forms. Pull-down experiments carried out with His-tagged forms, gel-filtration experiments and dimerization assays provided the first unequivocal experimental results for the role of the C-terminal residues in dimerization of the enzyme. There is a hydrophobic tunnel on the surface of HTLV-1 protease close to the C-terminal ends that is absent in the HIV-1 protease. This hydrophobic tunnel can accommodate the extra C-terminal residues of HTLV-1 protease, which was predicted to stabilize the dimer of the full-length enzyme and provides an alternative target site for protease inhibition.

Nuclear and nucleolar localization signals and their targeting function in phosphatidylinositol 4-kinase PI4K230.

PI4K230, an isoform of phosphatidylinositol 4-kinase, known primarily as a cytoplasmic membrane-bound enzyme, was detected recently also in the nucleolus of several cells. Here we provide mechanistic insight on the targeting function of its putative nuclear localization signal (NLS) sequences using molecular modeling, digitonin-permeabilized HeLa cells and binding to various importins. The synthetic sequence (916)NFNHIHKRIRRVADKYLSG(934) comprising a putative monopartite NLS (NLS1), targeted covalently bound fluorescent BSA to the nucleoplasm via classical importin alpha/beta mechanism employing importins alpha1 and alpha3 but not alpha5. This transport was inhibited by wheat germ agglutinin and GTPgammaS. The sequence (1414)SKKTNRGSQLHKYYMKRRTL(1433), a putative bipartite NLS (NLS2) proved ineffective in nuclear targeting if conjugated to fluorescently labeled BSA. Nonetheless, NLS2 or either of its basic clusters directed to the nucleolus soybean trypsin inhibitor that can pass the nuclear pore complex passively; moreover, an expressed 58 kDa fragment of PI4K230 (AA1166-1667) comprising NLS2 was also imported into the nucleus by import factors of reticulocyte lysate or by importin alpha1/beta or alpha3/beta complexes and localized to the nucleolus. We conclude that the putative bipartite NLS itself is a nucleolar targeting signal, and for nuclear import PI4K230 requires a larger sequence around it or, alternatively, the monopartite NLS.

Plasminogen activator inhibitor in human tears after laser refractive surgery.

PURPOSE: To observe levels of plasminogen activator inhibitor (PAI) in human tears after photorefractive keratectomy (PRK) and laser in situ keratomileusis (LASIK). SETTING: University medical center eye clinic. METHODS: Tear samples were collected from 46 eyes having PRK and 13 eyes having LASIK immediately before and after surgery and on the first (LASIK), third (PRK), and fifth (PRK) postoperative days. Analyses used enzyme-linked immunoassay, yielding 61 PRK PAI-1 determinations and 146 PRK and 35 LASIK PAI-2 determinations. RESULTS: All determinations of PRK PAI-1 were below the detection limit of 1 ng/mL of the original tear sample. In the PRK eyes, the mean PAI-2 concentration was 19.8 ng/mL +/- 23.4 (SD) in preoperative tears, 112.7 +/- 60.5 ng/mL immediately postoperatively, 12.1 +/- 19.5 ng/mL after 3 days, and 15.5 +/- 20.4 ng/mL after 5 days. In the LASIK eyes, the mean PAI-2 concentration was 19.0 +/- 33.1 ng/mL preoperatively, 111.5 +/- 69.2 ng/mL immediately postoperatively, and 15.7 +/- 18.8 ng/mL after 1 day. CONCLUSIONS: The similarity in the general time pattern of PAI-2 after PRK and LASIK suggests commonality in the enzymatic control response to corneal surgical wounding. Taken in the context of previous work, the observed levels of PAI-2 concentration in eyes with and without opacification suggest that in the postsurgical period, PAI-2 is not the controlling mechanism for the later development of corneal opacification and haze.

Novel macromolecular inhibitors of human immunodeficiency virus-1 protease.

An intracellularly expressed defective human immunodeficiency virus type-1 (HIV-1) protease (PR) monomer could function as a dominant-negative inhibitor of the enzyme that requires dimerization for activity. Based on in silico studies, two mutant PRs harboring hydrophilic mutations, capable of forming favorable inter- and intra-subunit interactions, were selected: PR(RE) containing Asp25Arg and Gly49Glu mutations, and PR(RER) containing an additional Ile50Arg mutation. The mutants were expressed and tested by PR assays, nuclear magnetic resonance (NMR) and cell culture experiments. The mutant PRs showed dose-dependent inhibition of the wild-type PR in a fluorescent microtiter plate PR assay. Furthermore, both mutants were retained by hexahistidine-tagged wild-type HIV-1 PR immobilized on nickel-chelate affinity resin. For the first time, heterodimerization between wild-type and dominant-negative mutant PRs were also demonstrated by NMR spectroscopy. (1)H-(15)N Heteronuclear Single Quantum Coherence NMR spectra showed that although PR(RE) has a high tendency to aggregate, PR(RER) exists mainly as a folded monomer at 25-35 microM concentration, but in the presence of wild-type PR in a ratio of 1:1, heterodimerization occurs with both mutants. While the recombinant virus containing the PR(RE) sequence showed only very low level of expression, expression of the viral proteins of the virus with the PR(RER) sequence was comparable with that of the wild-type. In cell culture experiments, infectivity of viral particles containing PR(RER) protein was reduced by 82%, at mutant to wild-type infective DNA ratio of 2:1.

Bovine leukemia virus protease: comparison with human T-lymphotropic virus and human immunodeficiency virus proteases.

Bovine leukemia virus (BLV) is a valuable model system for understanding human T-lymphotropic virus 1 (HTLV-1); the availability of an infectious BLV clone, together with animal-model systems, will help to explore anti-HTLV-1 strategies. Nevertheless, the specificity and inhibitor sensitivity of the BLV protease (PR) have not been characterized in detail. To facilitate such studies, a molecular model for the enzyme was built. The specificity of the BLV PR was studied with a set of oligopeptides representing naturally occurring cleavage sites in various retroviruses. Unlike HTLV-1 PR, but similar to the human immunodeficiency virus 1 (HIV-1) enzyme, BLV PR was able to hydrolyse the majority of the peptides, mostly at the same position as did their respective host PRs, indicating a broad specificity. When amino acid residues of the BLV PR substrate-binding sites were replaced by equivalent ones of the HIV-1 PR, many substitutions resulted in inactive protein, indicating a great sensitivity to mutations, as observed previously for the HTLV-1 PR. The specificity of the enzyme was studied further by using a series of peptides containing amino acid substitutions in a sequence representing a naturally occurring HTLV-1 PR cleavage site. Also, inhibitors of HIV-1 PR, HTLV-1 PR and other retroviral proteases were tested on the BLV PR. Interestingly, the BLV PR was more susceptible than the HTLV-1 PR to the inhibitors tested. Therefore, despite the specificity differences, in terms of mutation intolerance and inhibitor susceptibility of the PR, BLV and the corresponding animal-model systems may provide good models for testing of PR inhibitors that target HTLV-1.

Effect of experimental hypercholesterolaemia on K+ channel alpha-subunit mRNA levels in rabbit hearts.

We investigated the effect of dietary cholesterol on gene transcription of delayed rectifier (I(Kr) - ERG1 and I(Ks) - KvLQT1) and transient outward (I(to,fast) - Kv4.2 and Kv4.3) potassium channel subunits in rabbit hearts using real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR). While the level of Kv4.3 mRNA did not change, both Kv4.2 and ERG1 mRNAs were downregulated, whereas the level of KvLQT1 was increased in hypercholesterolaemic rabbits, indicating that hypercholesterolaemia altered ventricular K(+) channel alpha-subunit gene transcription.

Effect of mutations on the dimer stability and the pH optimum of the human foamy virus protease.

To explore the role of residues being close to the catalytic aspartates in the higher pH optimum and in the lower dimer stability of human foamy virus (HFV) protease (PR) in comparison with human immunodeficiency virus type 1 (HIV-1) protease, single (Q8R, H22L, S25T, T28D) and double (Q8R-T28D, H22L-T28D) mutants were created based on sequence alignments and on the molecular model of HFV PR. The wild-type and mutant enzymes were expressed in fusion with maltose binding protein in Escherichia coli and the fusion proteins were purified by affinity chromatography. Specificity constant of most mutants was lower, but the value of Q8R-T28D double mutant enzyme was higher than that of the wild-type HFV PR. Furthermore, urea denaturation at two pH values and pH optimum values showed an increased stability and pH optimum for most mutants. These results suggest that the mutated residues may not be responsible for the higher pH optimum of HFV PR, but they may contribute to the lower dimer stability as compared with that of HIV-1 PR.

Characterization of the murine leukemia virus protease and its comparison with the human immunodeficiency virus type 1 protease.

The protease (PR) of Murine leukemia virus (MLV) was expressed in Escherichia coli, purified to homogeneity and characterized by using various assay methods, including HPLC-based, photometric and fluorometric activity measurements. The specificity of the bacterially expressed PR was similar to that of virion-extracted PR. Compared with human immunodeficiency virus type 1 (HIV-1) PR, the pH optimum of the MLV enzyme was higher. The specificity of the MLV PR was further compared with that of HIV-1 PR by using various oligopeptides representing naturally occurring cleavage sites in MLV and HIV-1, as well as by using bacterially expressed proteins having part of the MLV Gag. Inhibitors designed against HIV-1 PR were also active on MLV PR, although all of the tested ones were substantially less potent on this enzyme than on HIV-1 PR. Nevertheless, amprenavir, the most potent inhibitor against MLV PR, was also able to block Gag processing in MLV-infected cells. These results indicate that, in spite of the similar function in the life cycle of virus infection, the two PRs are only distantly related in their specificity.

Improved purification protocol for wild-type and mutant human foamy virus proteases.

Wild-type and an active site mutant (S25T) human foamy virus (HFV) proteases were expressed in fusion with maltose binding protein in Escherichia coli. The mutant enzyme contained a Ser to Thr mutation in the -Asp-Ser-Gly- active site triplet of the enzyme, which forms the "fireman's grip" between the two subunits of the homodimeric enzyme. The fusion proteins were purified by affinity chromatography on amylose resin, cleaved with factor Xa, and the processed enzymes were purified by gel filtration under denaturing condition. Refolding after purification resulted in active enzymes with comparable yields. Furthermore, both enzymes showed similar catalytic activities in an oligopeptide substrate representing an HFV Gag cleavage site. However, the S25T mutant showed increased stability in urea unfolding experiment, in a good agreement with the suggested role of the Thr residue of fireman's grip.

Beta-lactam compounds as apparently uncompetitive inhibitors of HIV-1 protease.

Compounds of a combinatorial monocyclic beta-lactam library were found to be apparently uncompetitive inhibitors of HIV-1 protease, providing lead compounds for a new class of HIV protease inhibitors.