Analysis of a Novel Peptide That Is Capable of Inhibiting the Enzymatic Activity of the Protein Kinase A Catalytic Subunit-Like Protein from Trypanosoma equiperdum.

A 26-residue peptide possessing the αN-helix motif of the protein kinase A (PKA) regulatory subunit-like proteins from the Trypanozoom subgenera (VAP26, sequence = VAPYFEKSEDETALILKLLTYNVLFS), was shown to inhibit the enzymatic activity of the Trypanosoma equiperdum PKA catalytic subunit-like protein, in a similar manner that the mammalian heat-stable soluble PKA inhibitor known as PKI. However, VAP26 does not contain the PKI inhibitory sequence. Bioinformatics analyzes of the αN-helix motif from various Trypanozoon PKA regulatory subunit-like proteins suggested that the sequence could form favorable peptide-protein interactions of hydrophobic nature with the PKA catalytic subunit-like protein, which possibly may represent an alternative PKA inhibitory mechanism.

Disorder and partial folding in the regulatory subunit hinge region of Trypanosoma brucei protein kinase A: The C-linker portion inhibits the parasite's protein kinase A.

Microbial pathogens, such as Trypanosoma brucei, have an enormous impact on global health and economic systems. Protein kinase A of T. brucei is an attractive drug target as it is an essential enzyme which differs significantly from its human homolog.

Biochemical characterization of the cAMP-dependent protein kinase regulatory subunit-like protein from Trypanosoma equiperdum, detection of its inhibitory activity, and identification of potential interacting proteins.

An enriched fraction of an inhibitor of both the catalytic subunit of the cAMP-dependent protein kinase (PKA) from pig heart and a Trypanosoma equiperdum PKA catalytic subunit-like protein (TeqC-like) was obtained from the soluble fraction of T. equiperdum parasites after three consecutive purification steps: sedimentation through a linear 5-20% sucrose gradient, diethylaminoethyl-Sepharose anion-exchange chromatography, and Bio-Sil Sec-400-S size-exclusion high-performance liquid chromatography. The inhibitor was identified as the T.

Sequence Analysis of the cAMP-Dependent Protein Kinase Regulatory Subunit-Like Protein From Trypanosoma brucei.

Purpose: Study the N-terminal, C-terminal, and linker regions of the TbPKAr using homology modeling.

Methods: The amino acid sequences of the N-terminal, C-terminal, and linker regions of the TbPKAr were individually examined by means of BLAST analysis and in silico secondary structure predictions with several programs.

Results: The TbPKAr C-terminal region, showed a well-folded α/β structure, which consists of two concurrent flattened β-barrel-shaped domains that are separated by an elongated central α-helix similar to its mammalian counterpart, the TbPKAr linker region contains a PKA phosphorylation site and was predicted to be rather disordered.

Glucose deprivation activates a cAMP-independent protein kinase from Trypanosoma equiperdum.

Kemptide (sequence: LRRASLG) is a synthetic peptide holding the consensus recognition site for the catalytic subunit of the cAMP-dependent protein kinase (PKA). cAMP-independent protein kinases that phosphorylate kemptide were stimulated in Trypanosoma equiperdum following glucose deprivation. An enriched kemptide kinase-containing fraction was isolated from glucose-starved parasites using sedimentation throughout a sucrose gradient, followed by sequential chromatography on diethylaminoethyl-Sepharose and Sephacryl S-300.

Fluram-Kemptide-Lys8 Non-radioactive Assay for Protein Kinase A.

The cAMP-dependent protein kinase (PKA) is the best understood member of the superfamily of serine-threonine protein kinases and is involved in controlling a variety of cellular processes. Measurements of PKA activity traditionally relied on the use of [(32)P]-labeled ATP as the phosphate donor and a protein or peptide substrate as the phosphoaceptor. Recently non-isotopic assays for the PKA have been developed and this paper presents an improvement of a fluorometric assay for measuring the activity of PKA.

Immunological identification of a cAMP-dependent protein kinase regulatory subunit-like protein from the Trypanosoma equiperdum TeAp-N/D1 isolate.

Polyclonal immunoglobulin Y (IgY) antibodies were produced in chicken eggs against the purified R(II)-subunit of the cAMP-dependent protein kinase (PKA) from pig heart, which corresponds to the Sus scrofa R(II)α isoform. In order to evaluate whether Trypanosoma equiperdum possessed PKA R-like proteins, parasites from the Venezuelan TeAp-N/D1 strain were examined using the generated anti-R(II) IgY antibodies. Western blot experiments revealed a 57-kDa polypeptide band that was distinctively recognized by these antibodies.

Binding of rhodopsin and rhodopsin analogues to transducin, rhodopsin kinase and arrestin-1.

Aim: To investigate the interaction of reconstituted rhodopsin, 9-cis-retinal-rhodopsin and 13-cis-retinal-rhodopsin with transducin, rhodopsin kinase and arrestin-1.

Methods: Rod outer segments (ROS) were isolated from bovine retinas. Following bleaching of ROS membranes with hydroxylamine, rhodopsin and rhodopsin analogues were generated with the different retinal isomers and the concentration of the reconstituted pigments was calculated from their UV/visible absorption spectra.

Association of biomarkers with atherosclerosis and risk for coronary artery disease in patients with HIV.

Background: The massive use of Highly-Active Antiretroviral Therapy (HAART) in individuals with human immunodeficiency virus (HIV) coincided with an increase in cardiovascular disease, a major cause of morbidity and mortality in this group.

Objective: To determine the frequency of carotid atherosclerosis and the association between biomarker levels and carotid intimal-medial thickening in HIV-positive individuals treated for HIV at referral centers in Pernambuco.

Methods: This was a cross-sectional study of 122 HIV-positive patients.

Specific interactions of sticholysin I with model membranes: an NMR study.

Sticholysin I (StnI) is an actinoporin produced by the sea anemone Stichodactyla helianthus that binds biological and model membranes forming oligomeric pores. Both a surface cluster of aromatic rings and the N-terminal region are involved in pore formation. To characterize the membrane binding by StnI, we have studied by (1)H-NMR the environment of these regions in water and in the presence of membrane-mimicking micelles.