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Dopamine D2 receptor (D2R) is expressed in striatopallidal neurons and decreases forskolin-stimulated cyclic adenine monophosphate (cAMP) accumulation and gamma-aminobutyric acid (GABA) release. Dopamine D3 receptor (D3R) mRNA is expressed in a population of striatal D2R-expressing neurons. Also, D3R protein and binding have been reported in the neuropil of globus pallidus. We explore whether D2R and D3R colocalize in striatopallidal terminals and whether D3R modulates the D2R effect on forskolin-stimulated [3H]cAMP accumulation in pallidal synaptosomes and high K+ stimulated-[3H]GABA release in pallidal slices. Previous reports in heterologous systems indicate that calmodulin (CaM) and CaMKII modulate D2R and D3R functions; thus, we study whether this system regulates its functional interaction. D2R immunoprecipitates with CaM, and pretreatment with ophiobolin A or depolarization of synaptosomes with 15 mM of K+ decreases it. Both treatments increase the D2R inhibition of forskolin-stimulated [3H]cAMP accumulation when activated with quinpirole, indicating a negative modulation of CaM on D2R function. Quinpirole also activates D3R, potentiating D2R inhibition of cAMP accumulation in the ophiobolin A-treated synaptosomes. D2R and D3R immunoprecipitate in pallidal synaptosomes and decrease after the kainic acid striatal lesion, indicating the striatal origin of the presynaptic receptors. CaM-kinase II alfa (CaMKIIα) immunoprecipitates with D3R and increases after high K+ depolarization. In the presence of KN62, a CaMKIIα blocker, D3R potentiates D2R effects on cAMP accumulation in depolarized synaptosomes and GABA release in pallidal slices, indicating D3R function regulation by CaMKIIα. Our data indicate that D3R potentiates the D2R effect on cAMP accumulation and GABA release at pallidal terminals, an interaction regulated by the CaM-CaMKIIα system.

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Amyloid ß (Aß) oligomers are the most neurotoxic forms of Aß, and Aß(1-42) is the prevalent Aß peptide found in the amyloid plaques of Alzheimer's disease patients. Aß(25-35) is the shortest peptide that retains the toxicity of Aß(1-42). Aß oligomers bind to calmodulin (CaM) and calbindin-D28k with dissociation constants in the nanomolar Aß(1-42) concentration range. Aß and histidine-rich proteins have a high affinity for transition metal ions Cu2+, Fe3+ and Zn2+. In this work, we show that the fluorescence of Aß(1-42) HiLyteTM-Fluor555 can be used to monitor hexa-histidine peptide (His6) interaction with Aß(1-42). The formation of His6/Aß(1-42) complexes is also supported by docking results yielded by the MDockPeP Server. Also, we found that micromolar concentrations of His6 block the increase in the fluorescence of Aß(1-42) HiLyteTM-Fluor555 produced by its interaction with the proteins CaM and calbindin-D28k. In addition, we found that the His6-tag provides a high-affinity site for the binding of Aß(1-42) and Aß(25-35) peptides to the human recombinant cytochrome b5 reductase, and sensitizes this enzyme to inhibition by these peptides. In conclusion, our results suggest that a His6-tag could provide a valuable new tool to experimentally direct the action of neurotoxic Aß peptides toward selected cellular targets.

Assuntos

Assuntos

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The plasma membrane and autoinhibited Ca2+-ATPases contribute to the Ca2+ homeostasis in a wide variety of organisms. The enzymatic activity of these pumps is stimulated by calmodulin, which interacts with the target protein through the calmodulin-binding domain (CaMBD). Most information about this region is related to all calmodulin modulated proteins, which indicates general chemical properties and there is no established relation between Ca2+ pump sequences and taxonomic classification. Thus, the aim of this study was to perform an in silico analysis of the CaMBD from several Ca2+-ATPases, in order to determine their diversity and to detect specific patterns and amino acid selection in different species. Patterns related to potential and confirmed CaMBD were detected using sequences retrieved from the literature. The occurrence of these patterns was determined across 120 sequences from 17 taxonomical classes, which were analyzed by a phylogenetic tree to establish phylogenetic groups. Predicted physicochemical characteristics including hydropathy and net charge were calculated for each group of sequences. 22 Ca2+-ATPases sequences from animals, unicellular eukaryotes, and plants were retrieved from bioinformatic databases. These sequences allow us to establish the Patterns 1(GQILWVRGLTRLQTQ), 3(KNPSLEALQRW), and 4(SRWRRLQAEHVKK), which are present at the beginning of putative CaMBD of metazoan, parasites, and land plants. A pattern 2 (IRVVNAFR) was consistently found at the end of most analyzed sequences. The amino acid preference in the CaMBDs changed depending on the phylogenetic groups, with predominance of several aliphatic and charged residues, to confer amphiphilic properties. The results here displayed show a conserved mechanism to contribute to the Ca2+ homeostasis across evolution and may help to detect putative CaMBDs.

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In the present study, we reported the interactions at the molecular level of a series of compounds called Bisindolylmaleimide, as potential inhibitors of the calmodulin protein. Bisindolylmaleimide compounds are drug prototypes derived from Staurosporine, an alkaloid with activity for cancer treatment. Bisindolylmaleimide compounds II, IV, VII, X, and XI, are proposed and reported as possible inhibitors of calmodulin protein for the first time. For the above, a biotechnological device was used (fluorescent biosensor hCaM M124C-mBBr) to directly determine binding parameters experimentally (Kd and stoichiometry) of these compounds, and molecular modeling tools (Docking, Molecular Dynamics, and Chemoinformatic Analysis) to carry out the theoretical studies and complement the experimental data. The results indicate that this compound binds to calmodulin with a Kd between 193-248 nM, an order of magnitude lower than most classic inhibitors. On the other hand, the theoretical studies support the experimental results, obtaining an acceptable correlation between the ΔGExperimental and ΔGTheoretical (r2 = 0.703) and providing us with complementary molecular details of the interaction between the calmodulin protein and the Bisindolylmaleimide series. Chemoinformatic analyzes bring certainty to Bisindolylmaleimide compounds to address clinical steps in drug development. Thus, these results make these compounds attractive to be considered as possible prototypes of new calmodulin protein inhibitors.

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Melatonin (MEL) is a pleiotropic indolamine that reaches multiple intracellular targets. Among these, MEL binds to calmodulin (CaM) with high affinity. In presence of Ca2+, CaM binds to CaM-dependent kinase II (CaMKII). The Ca2+-CaM/CaMKII pathway regulates a myriad of brain functions in different cellular compartments. Evidence showing the regulation of this cellular pathway by MEL is scarce. Thus, our main objective was to study the interaction of MEL with CaM and its effects on CaMKII activity in two microenvironments (aqueous and lipidic) naturally occurring within the cell. In addition, colocalization of MEL with CaM in vivo was explored in mice brain hippocampus. In vitro CaM-MEL interaction and the structural conformations of CaM in the presence of this indoleamine were assessed through electrophoretic mobility and isoelectric point. The functional consequence of this interaction was evaluated by measuring CaMKII activity. Ca2+-CaM-MEL increased the activity of CaMKII in aqueous buffer but reduced the kinase activity in lipid buffer. Importantly, MEL colocalizes in vivo with Ca2+-CaM in the hippocampus. Our evidence suggests that MEL regulates the key cellular Ca2+-CaM/CaMKII pathway and might explain why physiological MEL concentrations reduce CaMKII activity in some experimental conditions, while in others it drives biological processes through activation of this kinase.

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Stress-related disorders display differences at multiple levels according to sex. While most studies have been conducted in male rodents, less is known about comparable outcomes in females. In this study, we found that the chronic restraint stress model (2.5 h/day for 14 days) triggers different somatic responses in male and female adult rats. Chronic restraint produced a loss in sucrose preference and novel location preference in male rats. However, chronic restraint failed to produce loss of sucrose preference in females, while it improved spatial performance. We then characterized the molecular responses associated with these behaviors in the hippocampus, comparing the dorsal and ventral poles. Notably, sex- and hippocampal pole-specific transcriptional signatures were observed, along with a significant concordance between the female ventral and male dorsal profiles. Functional enrichment analysis revealed both shared and specific terms associated with each pole and sex. By looking into signaling pathways that were associated with these terms, we found an ample array of sex differences in the dorsal and, to a lesser extent, in the ventral hippocampus. These differences were mainly present in synaptic TrkB signaling, Akt pathway, and glutamatergic receptors. Unexpectedly, the effects of stress on these pathways were rather minimal and mostly dissociated from the sex-specific behavioral outcomes. Our study suggests that female rats are resilient and males susceptible to the restraint stress exposure in the sucrose preference and object location tests, while the activity of canonical signaling pathways is primarily determined by sex rather than stress in the dorsal and ventral hippocampus.

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Metabolic syndrome is a pre-diabetic state characterized by several biochemical and physiological alterations, including insulin resistance, visceral fat accumulation, and dyslipidemias, which increase the risk for developing cardiovascular disease. Metabolic syndrome is associated with augmented sympathetic tone, which could account for the etiology of pre-diabetic cardiomyopathy. This review summarizes the current knowledge of the pathophysiological consequences of enhanced and sustained ß-adrenergic response in pre-diabetes, focusing on cardiac dysfunction reported in diet-induced experimental models of pre-diabetic cardiomyopathy. The research reviewed indicates that both protein kinase A and Ca2+/calmodulin-dependent protein kinase II play important roles in functional responses mediated by ß1-adrenoceptors; therefore, alterations in the expression or function of these kinases can be deleterious. This review also outlines recent information on the role of protein kinase A and Ca2+/calmodulin-dependent protein kinase II in abnormal Ca2+ handling by cardiomyocytes from diet-induced models of pre-diabetic cardiomyopathy.

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A study was performed on a collection of 84 isolates from decaying plant tissues and soils in Argentina previously identified as Trichoderma harzianum. Based on multiple phenotypic characters and multilocus phylogenetic analyses, 10 species were distinguished, three of which are described as new species: T. austroindianum, T. hortense, and T. syagri. Among the remaining seven identified species, the following five can be added to the Argentine mycobiota: T. afarasin, T. afroharzianum, T. endophyticum, T. guizhouense, and T. neotropicale. Trichoderma afroharzianum and T. endophyticum were the most frequent species found in the samples. In addition, a collection of isolates previously identified as T. harzianum with antagonistic abilities were reidentified as T. afroharzianum, thus highlighting the importance of correct identification of biocontrol species.

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Melocactus glaucescens is an endangered cactus highly valued for its ornamental properties. In vitro shoot production of this species provides a sustainable alternative to overharvesting from the wild; however, its propagation could be improved if the genetic regulation underlying its developmental processes were known. The present study generated de novo transcriptome data, describing in vitro shoot organogenesis induction in M. glaucescens. Total RNA was extracted from explants before (control) and after shoot organogenesis induction (treated). A total of 14,478 unigenes (average length, 520 bases) were obtained using Illumina HiSeq 3000 (Illumina Inc., San Diego, CA, USA) sequencing and transcriptome assembly. Filtering for differential expression yielded 2,058 unigenes. Pairwise comparison of treated vs. control genes revealed that 1,241 (60.3%) unigenes exhibited no significant change, 226 (11%) were downregulated, and 591 (28.7%) were upregulated. Based on database analysis, more transcription factor families and unigenes appeared to be upregulated in the treated samples than in controls. Expression of WOUND INDUCED DEDIFFERENTIATION 1 (WIND1) and CALMODULIN (CaM) genes, both of which were upregulated in treated samples, was further validated by real-time quantitative PCR (RT-qPCR). Differences in gene expression patterns between control and treated samples indicate substantial changes in the primary and secondary metabolism of M. glaucescens after the induction of shoot organogenesis. These results help to clarify the molecular genetics and functional genomic aspects underlying propagation in the Cactaceae family.