In view of the interest shown in phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P 2 ) as a second messenger, we studied the activation of protein kinase C␣ by this phosphoinositide. By using two double mutants from two different sites located in the C2 domain of protein kinase C␣, we have determined and characterized the PtdIns(4,5)P 2 -binding site in the protein, which was found to be important for its activation. Thus, there are two distinct sites in the C2 domain: the first, the lysinerich cluster located in the ␤3-and ␤4-sheets and which activates the enzyme through direct binding of PtdIns(4,5)P 2 ; and the second, the already well described site formed by the Ca 2؉ -binding region, which also binds phosphatidylserine and a result of which the enzyme is activated. The results obtained in this work point to a sequential activation model, in which protein kinase C␣ needs Ca 2؉ before the PtdIns(4,5)P 2 -dependent activation of the enzyme can occur.Phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P 2 ) 1 plays a key role in phosphoinositide signaling and regulates a wide range of processes at many subcellular sites. It is primarily detected in the plasma membrane but is also found in secretory vesicles, lysosomes, in the endoplasmic reticulum, the Golgi, and in the nucleus (1-5). PtdIns(4,5)P 2 can either bind to intracellular proteins and directly modulate their subcellular localization and activity, or it can act as a precursor for the generation of different second messengers. For example, several families of phospholipase C enzymes are responsible for the hydrolysis of PtdIns(4,5)P 2 in cells, leading to the production of diacylglycerol and inositol 1,4,5-trisphosphate (4, 6), which may, in turn, lead to the activation of different proteins such as some PKC isotypes.Protein kinase C (PKC) composes a large family of serine/ threonine kinases, which is activated by many extracellular signals and plays a critical role in many signal-transducing pathways in the cell (7-9). Based on their enzymatic properties, the mammalian PKC isotypes have been grouped into smaller subfamilies. The first group, which includes the classical isoforms ␣, ␤I, ␤II, and ␥, can be distinguished from the other groups because its activity is regulated by diacylglycerol (DAG) and, cooperatively, by Ca 2ϩ and acidic phospholipids, particularly phosphatidylserine (PS). Members of the second group are the novel mammalian (␦, ⑀, , and ) and yeast PKCs that are not regulated by Ca 2ϩ . The third group comprises the atypical PKC isoforms, , , and , whose regulation has not been clearly established, although it is clear that they are not regulated by DAG or Ca 2ϩ (8, 10). In classical PKC isoenzymes, Ca 2ϩ -dependent binding to membranes shows a high specificity for 1,2-sn-phosphatidyl-Lserine (11)(12)(13)(14). Additionally, this group of isoenzymes is sensitive to other anionic phospholipids, including phosphatidic acid and polyphosphoinositides (15-16) and to a variety of amphipathic membrane compounds, such as arachidonic acid and fre...

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Fourier transform infrared (FTIR) spectroscopy was used to investigate the structural and thermal denaturation of the C2 domain of PKC alpha (PKC-C2) and its complexes with Ca(2+) and phosphatidic acid vesicles. The amide I regions in the original spectra of PKC-C2 in the Ca(2+)-free and Ca(2+)-bound states are both consistent with a predominantly beta-sheet secondary structure below the denaturation temperatures. Spectroscopic studies of the thermal denaturation revealed that for the PKC-C2 domain alone the secondary structure abruptly changed at 50 degrees C. While in the presence of 2 and 12.5 mM Ca(2+), the thermal stability of the protein increased to 60 and 70 degrees C, respectively. Further studies using a mutant lacking two important amino acids involved in Ca(2+) binding (PKC-C2D246/248N) demonstrated that these mutations were inherently more stable to thermal denaturation than the wild-type protein. Phosphatidic acid binding to the PKC-C2 domain was characterized, and the lipid-protein binding became Ca(2+)-independent when 100 mol% phosphatidic acid vesicles were used. The mutant lacking two Ca(2+) binding sites was also able to bind to phosphatidic acid vesicles. The effect of lipid binding on secondary structure and thermal stability was also studied. Beta-sheet was the predominant structure observed in the lipid-bound state, although the percentage represented by this structure in the total area of the amide I band significantly decreased from 60% in the lipid-free state to 47% in the lipid-bound state. This decrease in the beta-sheet component of the lipid-bound complex correlates well with the significant increase observed in the 1644 cm(-1) band which can be assigned to loops and disordered structure. Thermal stability after lipid binding was very high, and no sign of thermal denaturation was observed in the presence of lipids under the conditions that were studied.

PKCepsilon is a member of the group of novel PKCs that contain a C2 domain located in their N-terminal region. On the basis of recent structural studies, a series of mutants were prepared to increase our knowledge of the mechanism of the phospholipid binding site of this domain. The results revealed that this domain preferentially binds to phosphatidic acid- and phosphatidylserine-containing vesicles. Although the increase in affinity was linear in the case of phosphatidic acid, it became exponential when the vesicles contained increasing concentrations of phosphatidylserine. Site-directed mutagenesis studies showed that residues W23, R26, and R32 located in loop 1 and I89 and Y91 located in loop 3 are of critical importance when the binding is performed with phosphatidic acid-containing vesicles. Furthermore, when the same mutants were assayed with phosphatidylserine-containing vesicles, no binding was observed in any case, reflecting the smaller affinity of the C2 domain for phosphatidylserine-containing vesicles. A study of the ionic nature of the membrane interaction suggested that it is mainly driven by electrostatic interactions that are disrupted by very low salt concentrations. Differential scanning calorimetry experiments performed to ascertain whether this interaction affected the transition phase of the phosphatidic acid demonstrated that increasing concentrations of the protein lead to changes in the transition, with more than one peak appearing at lower temperatures, which suggests a weak interaction focused on the polar headgroup of the phospholids. In conclusion, a different membrane-binding mode from those previously described in other C2 domains has been found and is seemingly based on electrostatic, interfacial, and hydrophobic interactions without the participation of Ca(2+) ions.

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Infrared spectroscopy (IR) and differential scanning calorimetry (DSC) were used to study the biophysical properties of the PKC:-C2 domain, a C2 domain that possess special characteristics as it binds to acidic phospholipids in a Ca 21 -independent manner and no structural information about it is available to date. When the secondary structure was determined by IR spectroscopy in H 2 O and D 2 O buffers, b sheet was seen to be the major structural component. Spectroscopic studies of the thermal denaturation in D 2 O showed a broadening in the amide I H band starting at 45 8C. Curve fitting analysis of the spectra demonstrated that two components appear upon thermal denaturation, one at 1623 cm 21 which was assigned to aggregation and a second one at 1645 cm 21 , which was assigned to unordered or open loop structures. A lipid binding assay has demonstrated that PKC:-C2 domain has preferencial affinity for PIP 2 although it exhibits maximal binding activity for phosphatidic acid when 100 mol% of this negatively charged phospholipid was used. Thus, phosphatidic acid containing vesicles were used to characterize the effect of lipid binding on the secondary structure and thermal stability. These experiments showed that the secondary structure did not change upon lipid binding and the thermal stability was very high with no significant changes occurring in the secondary structure after heating. DSC experiments demonstrated that when the C2-protein was scanned alone, it showed a T m of 49 8C and a calorimetric denaturation enthalpy of 144.318 kJ´mol 21 . However, when phoshatidic acid vesicles were included in the mixture, the transition disappeared and further IR experiments demonstrated that the protein structure was not modified under these conditions.

Background Chagas disease (CD) is a chronic parasitic disease caused by Trypanosoma cruzi and is endemic to continental Latin America. In Spain, the main transmission route is congenital. We aimed to assess adherence to regional recommendations of universal screening for CD during pregnancy in Latin American women in the province of Alicante from 2014 to 2018. Methodology/Principal findings Retrospective quality study using two availa sources: 1) delivery records of Latin American women that gave birth in the 10 public hospitals of Alicante between January 2014 and December 2018; and 2) records of Chagas serologies carried out in those centers between May 2013 and December 2018. There were 3026 deliveries in Latin American women during the study period; 1178 (38.9%) underwent CD serology. Screening adherence ranged from 17.2% to 59.3% in the different health departments and was higher in Bolivian women (48.3%). Twenty-six deliveries (2.2%) had a positive screening; CD was confirmed in 23 (2%) deliveries of 21 women. Bolivians had the highest seroprevalence (21/112; 18.7%), followed by Colombians (1/333; 0.3%) and Ecuadorians (1/348; 0.3%). Of 21 CD-positive women (19 Bolivians, 1 Colombian, 1 Ecuadorian), infection was already known in 12 (57.1%), and 9 (42.9%) had already been treated. Only 1 of the 12 untreated women (8.3%) was treated postpartum. Follow-up started in 20 of the 23 (87.0%) neonates but was completed only in 11 (47.8%); no cases of congenital transmission were detected. Among the 1848 unscreened deliveries, we estimate 43 undiagnosed cases of CD and 1 to 2 undetected cases of congenital transmission. Conclusions/Significance Adherence to recommendations of systematic screening for CD in Latin American pregnant women in Alicante can be improved. Strategies to strengthen treatment of postpartum women and monitoring of exposed newborns are needed. Currently, there may be undetected cases of congenital transmission in our province.

NIR offers multiple advantages for serum analysis, permitting a fast and direct determination of several parameters simultaneously, with low sample handling and without the need for reagents during the measurement step. The aim of this paper was to provide an evaluation of this technique in a real world scale, for the simultaneous determination of several parameters and based on a considerable number of samples. Direct near infrared (NIR) absorbance measurements were used to determine the concentration of clinical parameters in human serum that are required in routine biochemical tests. Total protein, albumin, total cholesterol, high-density lipoprotein (HDL cholesterol), low-density lipoprotein (LDL cholesterol), and very low-density lipoprotein (VLDL cholesterol), triglycerides, urea and glucose were determined in 447 serum samples obtained randomly from the clinical laboratory of the University Hospital Doctor Peset in Valencia (Spain). NIR spectra from 12 500 to 4000 cm À1 obtained with a 1 mm optical path length were evaluated by using partial least squares regression models (PLS) built from the spectra of samples with known concentrations provided by the hospital. Root mean square error crossvalidation (RMSECV) was used for selecting a number of factors, spectral regions and spectral preprocessing considered to build the models, that were evaluated from their prediction capability using the relative root mean square error of prediction (RRMSEP) of a series of around 30 independent samples, not used for calibration. For some analytes such as total protein, albumin, total cholesterol and triglycerides, errors obtained were 2.3, 4.4, 5.1, and 6.2% respectively, evidencing that the proposed methodology could compete with the enzymatic reference methodologies. However in the case of urea, glucose, HDL and LDL, average errors obtained were 16.0, 16.2, 18.0 and 11.0% respectively, and therefore the NIR methodology proposed is limited as a screening tool. With the use of a considerable number of samples for calibration, this study confirms that the proposed green and cost-effective methodology is ready for scaling up from the bench to the real world.