Structural modeling and functional analysis of the essential ribosomal processing protease Prp from Staphylococcus aureus

In Firmicutes and related bacteria, ribosomal large subunit protein L27 is encoded with a conserved N-terminal extension that is removed to expose residues critical for ribosome function. Bacteria encoding L27 with this N-terminal extension also encode a sequence-specific cysteine protease, Prp, which carries out this cleavage. In this work, we demonstrate that L27 variants with an un-cleavable N-terminal extension, or lacking the extension (pre-cleaved), are unable to complement an L27 deletion in Staphylococcus aureus. This indicates that N-terminal processing of L27 is not only essential but possibly has a regulatory role. Prp represents a new clade of previously uncharacterized cysteine proteases, and the dependence of S. aureus on L27 cleavage by Prp validates the enzyme as a target for potential antibiotic development. To better understand the mechanism of Prp activity, we analyzed Prp enzyme kinetics and substrate preference using a fluorogenic peptide cleavage assay. Molecular modeling and site-directed mutagenesis implicate several residues around the active site in catalysis and substrate binding, and support a structural model in which rearrangement of a flexible loop upon binding of the correct peptide substrate is required for the active site to assume the proper conformation. These findings lay the foundation for the development of antimicrobials that target this novel, essential pathway

Kinetics and Inhibition Studies of the L205R Mutant of cAMP-Dependent Protein Kinase Involved in Cushing’s Syndrome

Overproduction of cortisol by the hypothalamus–pituitary–adrenal hormone system results in the clinical disorder known as Cushing\u27s syndrome. Genomics studies have identified a key mutation (L205R) in the α‐isoform of the catalytic subunit of cAMP‐dependent protein kinase (PKACα) in adrenal adenomas of patients with adrenocorticotropic hormone‐independent Cushing\u27s syndrome. Here, we conducted kinetics and inhibition studies on the L205R‐PKACα mutant. We have found that the L205R mutation affects the kinetics of both Kemptide and ATP as substrates, decreasing the catalytic efficiency (kcat/KM) for each substrate by 12‐fold and 4.5‐fold, respectively. We have also determined the IC50 and Ki for the peptide substrate‐competitive inhibitor PKI(5–24) and the ATP‐competitive inhibitor H89. The L205R mutation had no effect on the potency of H89, but causes a \u3e 250‐fold loss in potency for PKI(5–24). Collectively, these data provide insights for the development of L205R‐PKACα inhibitors as potential therapeutics

Study of the Putative Fusion Regions of the preS Domain of Hepatitis B Virus

In a previous study, it was shown that purified preS domains of hepatitis B virus (HBV) could interact with acidic phospholipid vesicles and induce aggregation, lipid mixing and leakage of internal contents which could be indicative of their involvement in the fusion of the viral and cellular membranes (Núñez, E. et al. 2009. Interaction of preS domains of hepatitis B virus with phospholipid vesicles. Biochim. Biophys. Acta 17884:417-424). In order to locate the region responsible for the fusogenic properties of preS, five mutant proteins have been obtained from the preS1 domain of HBV, in which 40 amino acids have been deleted from the sequence, with the starting point of each deletion moving 20 residues along the sequence. These proteins have been characterized by fluorescence and circular dichroism spectroscopy, establishing that, in all cases, they retain their mostly non-ordered conformation with a high percentage of β structure typical of the full-length protein. All the mutants can insert into the lipid matrix of dimyristoylphosphatidylglycerol vesicles. Moreover, we have studied the interaction of the proteins with acidic phospholipid vesicles and each one produces, to a greater or lesser extent, the effects of destabilizing vesicles observed with the full-length preS domain. The ability of all mutants, which cover the complete sequence of preS1, to destabilize the phospholipid bilayers points to a three-dimensional structure and/or distribution of amino acids rather than to a particular amino acid sequence as being responsible for the membrane fusion process

Circular dichroism and fluorescence spectroscopic properties of the major core protein of feline immunodeficiency virus and its tryptophan mutants: Assignment of the individual contribution of the aromatic side chains

The gene coding for the major capsid protein of feline immunodeficiency virus (FIV) has been cloned into the expression vector pQE60, which allows protein purification by affinity chromatography on a nitrilotriacetic acid/Ni/agarose column. The gene was expressed in Escherichia coli and the resultant soluble protein (FIV-rp24) purified to electrophoretic homogeneity. The amino-acid composition of the recombinant protein is almost identical to that predicted from the DNA sequence. This protein has two tryptophan residues at positions 40 and 126 that have been replaced by phenylalanine by site-directed mutagenesis to obtain two single mutants and a double mutant. Circular dichroism and fluorescence spectroscopy were employed to study the structural features of FIV-rp24 protein and its tryptophan mutants. The analysis of the CD spectra indicated that α-helix is the major secondary structural element (48-52%) and that the overall three-dimensional structure is not modified by the mutations. The fluorescence emission spectra showed that both tryptophan residues occupy a highly hydrophobic environment. Moreover, the different tyrosine fluorescence intensities of wild-type and mutant proteins are indicative of the existence of resonance energy transfer processes to nearby tryptophan. The individual contributions of each tryptophan residue to the spectroscopic properties of the wild-type protein were obtained from the spectra of all these proteins. Thermal denaturation studies indicate that the two tryptophan residues do not contribute equally to the stabilization of the three-dimensional structure

Expression and structural properties of a chimeric protein based on the ectodomains of E1 and E2 hepatitis C virus envelope glycoproteins

Hepatitis C virus encodes two enveloped glycoproteins, E1 and E2, which are involved in viral attachment and entry into target cells. We have obtained in insect cells infected by recombinant baculovirus a chimeric secreted recombinant protein, E1341E2661, containing the ectodomains of E1 and E2. The described procedure allows the purification of approximately 2 mg of protein from 1 L of culture media. Sedimentation velocity experiments and SDS-PAGE in the absence of reducing agents indicate that the protein has a high tendency to self-associate, the dimer being the main species observed. All the oligomeric forms observed maintain a conformation which is recognized by the conformation-dependent monoclonal antibody H53 directed against the E2 ectodomain. The spectroscopic properties of E1341E2661 are those of a three-dimensionally structured protein. Moreover, the chimeric protein is able to bind to human antibodies present in HCV-positive human sera. Accordingly, this chimeric soluble polypeptide chain may be a valuable tool to study the structure-function relationship of HCV envelope proteins

Fusogenic properties of the ectodomains of hepatitis C virus envelope proteins

We have used an isolated chimeric protein E1340E2661 that includes the ectodomains of the envelope proteins of epatitis C virus to study its interaction with model membranes. E1340E2661 has some of the membrane destabilization properties, vesicle aggregation, lipid mixing and the release of internal aqueous content, which have previously been ascribed to fusion proteins. The effects are preferentially produced on vesicles of acidic phospholipids which would indicate the importance of the electrostatic interactions. In fact, an increase of the ionic strength of the buffer induced a considerable decrease of the destabilizing properties. Moreover, fluorescence polarization studies show that the recombinant protein reduces the amplitude of the thermal transition of dimyristoylphosphatidylglycerol vesicles and increases the transition temperature at pH 5.0 in a dose-dependent manner, indicating its insertion into the bilayer. Furthermore, a decrease of the pH induces a onformational change in the protein structure as videnced by fluorescence of tryptophan residues and 4,4-bis(1-anilinonaphthalene-8-sulfonate). A model for the fusion of hepatitis C virus with the host cell membrane can be postulated. The dissociation of E1E2 dimers would uncover the fusion peptides which can then interact with the polar lipid heads of the outer leaflet of the lipid bilayer and next insert into the hydrophobic moiety producing the destabilization of the bilayer which finally leads to fusion

Winter grazing of corn residues: Effects on soil properties and subsequent crop yields from a corn-soybean crop rotation

Corn residues could be a good resource for winter cattle grazing. The study investigates whether winter grazing causes soil compaction and yield reduction in crops that are planted following grazing

Effects of Corn Crop Residue Grazing on Soil Physical Properties and Subsequent Soybean Production in a Corn-Soybean Crop Rotation (A Progress Report)

The highest cost to beef cow-calf producers is the feeding of stored feeds in winter months. To lower feed costs, many producers will try to extend the grazing season into the winter. The primary resource for winter grazing in the Midwest is corn crop residues. On the average, corn crop residue grazing will reduce the amount of hay needed to maintain cows by approximately one-half ton per acre grazed over the winter. Although crop residue grazing is quite effective in reducing feed costs, some producers are concerned that corn residue grazing will have an adverse effect on soybean yields the following year resulting from soil compaction. It has already been proven that the use of large machinery will cause soil compaction in wet conditions and that it reduces corn grain yields from 6 to 10%

Effects of Corn Crop Residue Grazing on Soil Physical Properties and Subsequent Soybean Production in a Corn–Soybean Crop Rotation (A Progress Report)

Beginning in 1999, two locations in Iowa (Chariton, Atlantic) were used to study the effects of corn residue grazing by beef cows on soil characteristics and soybean yields the following growing season. Cows were allowed to graze inside selected paddocks at monthly periods throughout the fall and winter. For a grazed and ungrazed comparison, grazing exclosures were used inside the grazed paddocks, while one paddock was left ungrazed for a control. Also, the following year, equal portions of the fields went to no-tillage and disked soil prior to soybean planting so that effects of corn residue grazing on tillage treatments could be compared. The use of this design was to determine whether grazing had adverse effects on soil characteristics and, if so, at what date and weather conditions they occurred. Soil was analyzed for soil bulk density, moisture, penetration resistance, roughness, texture, and type. Corn crop residues were collected for yield, cover, and composition. Precipitation and soil temperature also were recorded throughout the grazing season. The following year, soybeans were harvested using a combine equipped with a yield monitor and global positioning system (GPS). After two years of study at both locations, some grazing fields with corn crop residue have shown effects on soil and crop residue characteristics. Organic matter (OM) yield of crop residue generally decreases at the faster rate in grazed fields than organic matter of ungrazed fields. However, corn crop residue composition was the same in grazed and ungrazed fields except for the 1999-2000 season at Chariton where crude protein decreased but acid detergent insoluble nitrogen (ADIN) increased with no difference in fiber content between grazed and ungrazed paddocks. Corn crop residue cover and soil roughness both can be greatly affected by the interaction of grazing and weather conditions. When the temperature is above freezing and precipitation is adequate, cattle traffic can cause roughness, while reducing residue cover by working it into the soil. Even though grazing corn residue by cattle can increase the surface roughness, it has not yet caused any increase in bulk density measurements or any reduction in soybean yields. Penetration resistance ratios have shown some significant difference between grazed and ungrazed paddocks, but the reason is unclear

Anticorpos muscarínicos e resposta da frequência cardíaca ao exercício dinâmico e a manobra de Valsalva na doença de Chagas crônica

We have studied the cardiac chronotropic responses to the Valsalva maneuver and to dynamic exercise of twenty chronic chagasic patients with normal left ventricular function and no segmental wall abnormalities by two-dimensional echocardiogram. The absolute increase in heart rate of the patients (Δ = 21.5 ± 10 bpm, M±SD) during the maneuver was significantly diminished when compared to controls (Δ = 31.30 ± 70, M±SD, p = 0.03). The minimum heart rate (58.24 ± 8.90 vs. 62.80 ± 10, p = 0.68) and the absolute decrease in heart rate at the end of the maneuver (Δ = 38.30 ± 13 vs. Δ = 31.47 ± 17, p = 0.10) were not different from controls. The initial heart rate acceleration during dynamic exercise (Δ = 12 ± 7.55 vs. Δ = 19 ± 7.27, M±SD, p = 0.01) was also diminished, but the heart rate recovery during the first ten seconds was more prominent in the sero-positive patients (Median: 14, Interquartile range: (9.75-17.50 vs. 5(0-8.75, p = 0.001). The serum levels of muscarinic cardiac auto-antibodies were significantly higher in the chagasic patients (Median: 34.58, Interquartile Range: 17-46.5, Optical Density) than in controls (Median: 0, Interquartile Range: 0-22.25, p = 0.001) and correlated significantly and directly (r = 0.68, p = 0.002) with early heart rate recovery during dynamic exercise. The results of this investigation indirectly suggest that, the cardiac muscarinic auto-antibodies may have positive agonist effects on parasympathetic heart rate control of chagasic patients.Foram estudadas as respostas cronotrópicas cardíacas à manobra de Valsalva e ao exercício dinâmico de vinte pacientes chagásicos com função ventricular esquerda normal e sem alterações da contractilidade segmentar por ecocardiografia bidimensional. O aumento absoluto da frequência cardíaca dos pacientes (Δ = 21,5 ± 10 bpm, M ± DP) durante a manobra de Valsalva foi significativamente menor quando se comparava ao grupo controle (Δ = 31,30 ± 70, p = 0,03). A frequência cardíaca mínima (58,24 ± 8,90 vs 62,80 ± 10, p = 0,68) e a diminuição da frequência cardíaca absoluta no final da manobra (Δ = 38,30 ± 13 vs Δ = 31,47 ± 17, p = 0,10) não foram diferentes em comparação com o grupo controle. A aceleração inicial da frequência cardíaca durante o exercício dinâmico (Δ = 12 ± 7,55 vs Δ = 19 ± 7,27, p = 0,01) também foi menor, mas a recuperação da frequência cardíaca, durante os primeiros dez segundos, foi maior no grupo sero-positivos [mediana:14 (intervalo interquartil: 9,75-17,50) vs 5 (0 - 8,75), p = 0,001]. Os níveis séricos de auto-anticorpos muscarínicos cardíacos foram significativamente maiores nos pacientes chagásicos do que no grupo controle [(mediana: 34,58 densidade óptica (intervalo interquartil 17 - 46,5) vs (mediana: 0, intervalo interquartil 0 - 22,25) p = 0,001] e a correlação é significativa e direta (r = 0,68, p = 0,002) com o início da recuperação da frequência cardíaca durante o exercício dinâmico. Os resultados desta investigação sugerem que indiretamente, os auto-anticorpos muscarínicos cardíacos, podem ter ação agonista positiva sobre o controle parassimpático da frequência cardíaca dos pacientes chagásicos