The vnd/NK-2 homeodomain: thermodynamics of reversible unfolding and DNA binding for wild-type and with residue replacements H52R and H52R/T56W in helix III

The conformational stabilities of the vnd (ventral nervous system defective)/NK-2 homeodomain [HD(wt); residues 1-80 that encompass the 60-residue homeodomain] and those harboring mutations in helix III of the DNA recognition site [HD(H52R) and HD(H52R/T56W)] have been investigated by differential scanning calorimetry (DSC) and ellipticity changes at 222 nm. Thermal unfolding reactions at pH 7.4 are reversible and repeatable in the presence of 50-500 mM NaCl with DeltaC(p) = 0.52 +/- 0.04 kcal K(-1) mol(-1). A substantial stabilization of HD(wt) is produced by 50 mM phosphate or by the addition of 100-500 mM NaCl to 50 mM Hepes, pH 7.4, buffer (from T(m) = 35.5 degrees C to T(m) 43-51 degrees C; DeltaH(vH) congruent with 47 +/- 5 kcal mol(-1)). The order of stability is HD(H52R/T56W) > HD(H52R) > HD(wt), irrespective of the anions present. Progress curves for ellipticity changes at 222 nm as a function of increasing temperature are fitted well by a two-state unfolding model, and the cooperativity of secondary structure changes is greater for mutant homeodomains than for HD(wt) and also is increased by adding 100 mM NaCl to Hepes buffer. A 33% quench of the intrinsic tryptophanyl residue fluorescence of HD(wt) by phosphate binding (K(D)' = 2.6 +/- 0.3 mM phosphate) is reversed approximately 60% by DNA binding. Thermodynamic parameters for vnd/NK-2 homeodomain proteins binding sequence-specific 18 bp DNA have been determined by isothermal titration calorimetry (10-30 degrees C). Values of DeltaC(p) are +0.25, -0.17, and -0.10 +/- 0.04 kcal K(-1) mol(-1) for HD(wt), HD(H52R), and HD(H52R/T56W) binding duplex DNA, respectively. Interactions of homeodomains with DNA are enthalpically controlled at 298 K and pH 7.4 with corresponding DeltaH values of -6.6 +/- 0.5, -10.8 +/- 0.1, and -9.0 +/- 0.6 kcal mol(-1) and DeltaG' values of -11.0 +/- 0.1, -11.0 +/- 0.1, and -11.3 +/- 0.3 kcal mol(-1) with a binding stoichiometry of 1.0 +/- 0.1. Thermodynamic parameters for DNA binding are not predicted from homeodomain structural changes that occur upon complexing to DNA and must reflect also solvent and possibly DNA rearrangements.     

Shorter leukocyte telomere length is associated with severity of COVID-19 infection.

The infection by COVID-19 is a serious global public health problem. An efficient way to improve this disease's clinical management would be to characterize patients at higher risk of progressing to critically severe infection using prognostic biomarkers. The telomere length could be used for this purpose. Telomeres are responsible for controlling the number of maximum cell divisions. The telomere length is a biomarker of aging and several diseases. We aimed to compare leukocyte telomere length (LTL) between patients without COVID-19 and patients with different clinical severity of the infection. Were included 53 patients who underwent SARS-CoV-2 PCR divided in four groups. The first group was composed by patients with a negative diagnosis for COVID-19 (n = 12). The other three groups consisted of patients with a confirmed diagnosis of COVID-19 divided according to the severity of the disease: mild (n = 15), moderate (n = 17) and severe (n = 9). The LTL was determined by Q-PCR. The severe group had the shortest LTL, followed by the moderate group. The negative and mild groups showed no differences. There is an increase of patients with hypertension (p = 0.0099) and diabetes (p = 0.0067) in moderate and severe groups. Severe group was composed by older patients in comparison with the other three groups (p = 0.0083). Regarding sex, there was no significant difference between groups (p = 0.6279). In an ordinal regression model, only LTL and diabetes were significantly associated with disease severity. Shorter telomere length was significantly associated with the severity of COVID-19 infection, which can be useful as a biomarker or to better understand the SARS-CoV-2 pathophysiology.     

Exocyst function is regulated by effector phosphorylation

The exocyst complex tethers vesicles at sites of fusion through interactions with small GTPases. The G protein RalA resides on Glut4 vesicles, and binds to the exocyst after activation by insulin, but must then disengage to ensure continuous exocytosis. Here we report that, after recognition of the exocyst by activated RalA, disengagement occurs through phosphorylation of its effector Sec5, rather than RalA inactivation. Sec5 undergoes phosphorylation in the G-protein binding domain, allosterically reducing RalA interaction. The phosphorylation event is catalysed by protein kinase C and is reversed by an exocyst-associated phosphatase. Introduction of Sec5 bearing mutations of the phosphorylation site to either alanine or aspartate disrupts insulin-stimulated Glut4 exocytosis, as well as other trafficking processes in polarized epithelial cells and during development of zebrafish embryos. The exocyst thus serves as a 'gatekeeper' for exocytic vesicles through a circuit of engagement, disengagement and re-engagement with G proteins.     

A call for using natural compounds in the development of new antimalarial treatments - an introduction

Natural compounds, mostly from plants, have been the mainstay of traditional medicine for thousands of years. They have also been the source of lead compounds for modern medicine, but the extent of mining of natural compounds for such leads decreased during the second half of the 20th century. The advantage of natural compounds for the development of drugs derives from their innate affinity for biological receptors. Natural compounds have provided the best anti-malarials known to date. Recent surveys have identified many extracts of various organisms (mostly plants) as having antiplasmodial activity. Huge libraries of fractionated natural compounds have been screened with impressive hit rates. Importantly, many cases are known where the crude biological extract is more efficient pharmacologically than the most active purified compound from this extract. This could be due to synergism with other compounds present in the extract, that as such have no pharmacological activity. Indeed, such compounds are best screened by cell-based assay where all potential targets in the cell are probed and possible synergies identified. Traditional medicine uses crude extracts. These have often been shown to provide many concoctions that deal better with the overall disease condition than with the causative agent itself. Traditional medicines are used by ~80 % of Africans as a first response to ailment. Many of the traditional medicines have demonstrable anti-plasmodial activities. It is suggested that rigorous evaluation of traditional medicines involving controlled clinical trials in parallel with agronomical development for more reproducible levels of active compounds could improve the availability of drugs at an acceptable cost and a source of income in malaria endemic countries.