Parathymosin affects the binding of linker histone H1 to nucleosomes and remodels chromatin structure

Linker histone H1 is the major factor that stabilizes higher order chromatin structure and modulates the action of chromatin-remodeling enzymes. We have previously shown that parathymosin, an acidic, nuclear protein binds to histone H1 in vitro and in vivo. Confocal laser scanning microscopy reveals a nuclear punctuate staining of the endogenous protein in interphase cells, which is excluded from dense heterochromatic regions. Using an in vitro chromatin reconstitution system under physiological conditions, we show here that parathymosin (ParaT) inhibits the binding of H1 to chromatin in a dose-dependent manner. Consistent with these findings, H1-containing chromatin assembled in the presence of ParaT has reduced nucleosome spacing. These observations suggest that interaction of the two proteins might result in a conformational change of H1. Fluorescence spectroscopy and circular dichroism-based measurements on mixtures of H1 and ParaT confirm this hypothesis. Human sperm nuclei challenged with ParaT become highly decondensed, whereas overexpression of green fluorescent protein- or FLAG-tagged protein in HeLa cells induces global chromatin decondensation and increases the accessibility of chromatin to micrococcal nuclease digestion. Our data suggest a role of parathymosin in the remodeling of higher order chromatin structure through modulation of H1 interaction with nucleosomes and point to its involvement in chromatin-dependent functions.     

Ser and Thr Residues Modulate the Conformation of Pro-Kinked Transmembrane α-Helices

Functionally required conformational plasticity of transmembrane proteins implies that specific structural motifs have been integrated in transmembrane helices. Surveying a database of transmembrane helices and the large family of G-protein coupled receptors we identified a series of overrepresented motifs associating Pro with either Ser or Thr. Thus, we have studied the conformation of Pro-kinked transmembrane helices containing Ser or Thr residues, in both g+ and g− rotamers, by molecular dynamics simulations in a hydrophobic environment. Analysis of the simulations shows that Ser or Thr can significantly modulate the deformation of the Pro. A series of motifs, such as (S/T)P and (S/T)AP in the g+ rotamer and the TAP and PAA(S/T) motifs in the g− rotamer, induce an increase in bending angle of the helix compared to a standard Pro-kink, apparently due to the additional hydrogen bond formed between the side chain of Ser/Thr and the backbone carbonyl oxygen. In contrast, (S/T)AAP and PA(S/T) motifs, in both g+ and g−, and PAA(S/T) in g+ rotamers decrease the bending angle of the helix by either reducing the steric clash between the pyrrolidine ring of Pro and the helical backbone, or by adding a constrain in the form of a hydrogen bond in the curved-in face of the helix. Together with a number of available experimental data, our results strongly suggest that association of Ser and Thr with Pro is commonly used in transmembrane helices to accommodate the structural needs of specific functions.     

Differential Function of Lip Residues in the Mechanism and Biology of an Anthrax Hemophore

To replicate in mammalian hosts, bacterial pathogens must acquire iron. The majority of iron is coordinated to the protoporphyrin ring of heme, which is further bound to hemoglobin. Pathogenic bacteria utilize secreted hemophores to acquire heme from heme sources such as hemoglobin. Bacillus anthracis, the causative agent of anthrax disease, secretes two hemophores, IsdX1 and IsdX2, to acquire heme from host hemoglobin and enhance bacterial replication in iron-starved environments. Both proteins contain NEAr-iron Transporter (NEAT) domains, a conserved protein module that functions in heme acquisition in Gram-positive pathogens. Here, we report the structure of IsdX1, the first of a Gram-positive hemophore, with and without bound heme. Overall, IsdX1 forms an immunoglobin-like fold that contains, similar to other NEAT proteins, a 3₁₀-helix near the heme-binding site. Because the mechanistic function of this helix in NEAT proteins is not yet defined, we focused on the contribution of this region to hemophore and NEAT protein activity, both biochemically and biologically in cultured cells. Site-directed mutagenesis of amino acids in and adjacent to the helix identified residues important for heme and hemoglobin association, with some mutations affecting both properties and other mutations affecting only heme stabilization. IsdX1 with mutations that reduced the ability to associate with hemoglobin and bind heme failed to restore the growth of a hemophore-deficient strain of B. anthracis on hemoglobin as the sole iron source. These data indicate that not only is the 3₁₀-helix important for NEAT protein biology, but also that the processes of hemoglobin and heme binding can be both separate as well as coupled, the latter function being necessary for maximal heme-scavenging activity. These studies enhance our understanding of NEAT domain and hemophore function and set the stage for structure-based inhibitor design to block NEAT domain interaction with upstream ligands.     

Glycine-alanine repeats impair proper substrate unfolding by the proteasome

Proteasome ATPases unravel folded proteins. Introducing a sequence containing only glycine and alanine residues (GAr) into substrates can impair their digestion. We previously proposed that a GAr interferes with the unfolding capacity of the proteasome, leading to partial degradation of products. Here we tested that idea in several ways. Stabilizing or destabilizing a folded domain within substrate proteins changed GAr-mediated intermediate production in the way predicted by the model. A downstream folded domain determined the sites of terminal proteolysis. The spacing between a GAr and a folded domain was critical for intermediate production. Intermediates containing a GAr did not remain associated with proteasomes, excluding models whereby retained GAr-containing proteins halt further processing. The following model is supported: a GAr positioned within the ATPase ring reduces the efficiency of coupling between nucleotide hydrolysis and work performed on the substrate. If this impairment takes place when unfolding must be initiated, insertion pauses and proteolysis is limited to the portion of the substrate that has already entered the catalytic chamber of the proteasome.     

Vitamin D and Caudal Primary Motor Cortex: A Magnetic Resonance Spectroscopy Study

Background

Vitamin D is involved in brain physiology and lower-extremity function. We investigated spectroscopy in a cohort of older adults to explore the hypothesis that lower vitamin D status was associated with impaired neuronal function in caudal primary motor cortex (cPMC) measured by proton magnetic resonance spectroscopic imaging.

Methods

Twenty Caucasian community-dwellers (mean±standard deviation, 74.6±6.2 years; 35.0% female) from the ‘Gait and Brain Study’ were included in this analysis. Ratio of N-acetyl-aspartate to creatine (NAA/Cr), a marker of neuronal function, was calculated in cPMC. Participants were categorized according to mean NAA/Cr. Lower vitamin D status was defined as serum 25-hydroxyvitamin D (25OHD) concentration <75 nmol/L. Age, gender, number of comorbidities, vascular risk, cognition, gait performance, vitamin D supplements, undernourishment, cPMC thickness, white matter hyperintensities grade, serum parathyroid hormone concentration, and season of evaluation were used as potential confounders.

Results

Compared to participants with high NAA/Cr (n = 11), those with low NAA/Cr (i.e., reduced neuronal function) had lower serum 25OHD concentration (P = 0.044) and more frequently lower vitamin D status (P = 0.038). Lower vitamin D status was cross-sectionally associated with a decrease in NAA/Cr after adjustment for clinical characteristics (β = −0.41, P = 0.047), neuroimaging measures (β = −0.47, P = 0.032) and serum measures (β = −0.45, P = 0.046).

Conclusions

Lower vitamin D status was associated with reduced neuronal function in cPMC. These novel findings need to be replicated in larger and preferably longitudinal cohorts. They contribute to explain the pathophysiology of gait disorders in older adults with lower vitamin D status, and provide a scientific base for vitamin D replacement trials.     

Waixenicin A inhibits cell proliferation through magnesium-dependent block of transient receptor potential melastatin 7 (TRPM7) channels

Transient receptor potential melastatin 7 (TRPM7) channels represent the major magnesium-uptake mechanism in mammalian cells and are key regulators of cell growth and proliferation. They are expressed abundantly in a variety of human carcinoma cells controlling survival, growth, and migration. These characteristics are the basis for recent interest in the channel as a target for cancer therapeutics. We screened a chemical library of marine organism-derived extracts and identified waixenicin A from the soft coral Sarcothelia edmondsoni as a strong inhibitor of overexpressed and native TRPM7. Waixenicin A activity was cytosolic and potentiated by intracellular free magnesium (Mg(2+)) concentration. Mutating a Mg(2+) binding site on the TRPM7 kinase domain reduced the potency of the compound, whereas kinase deletion enhanced its efficacy independent of Mg(2+). Waixenicin A failed to inhibit the closely homologous TRPM6 channel and did not significantly affect TRPM2, TRPM4, and Ca(2+) release-activated Ca(2+) current channels. Therefore, waixenicin A represents the first potent and relatively specific inhibitor of TRPM7 ion channels. Consistent with TRPM7 inhibition, the compound blocked cell proliferation in human Jurkat T-cells and rat basophilic leukemia cells. Based on the ability of the compound to inhibit cell proliferation through Mg(2+)-dependent block of TRPM7, waixenicin A, or structural analogs may have cancer-specific therapeutic potential, particularly because certain cancers accumulate cytosolic Mg(2+).     

Serum 25-Hydroxyvitamin D Concentrations ≥40 ng/ml Are Associated with >65% Lower Cancer Risk: Pooled Analysis of Randomized Trial and Prospective Cohort Study

BackgroundHigher serum 25-hydroxyvitamin D [25(OH)D] concentrations have been associated with a lower risk of multiple cancer types across a range of 25(OH)D concentrations.ObjectivesTo investigate whether the previously reported inverse association between 25(OH)D and cancer risk could be replicated, and if a 25(OH)D response region could be identified among women aged 55 years and older across a broad range of 25(OH)D concentrations.MethodsData from two cohorts representing different median 25(OH)D concentrations were pooled to afford a broader range of 25(OH)D concentrations than either cohort alone: the Lappe cohort (N = 1,169), a randomized clinical trial cohort (median 25(OH)D = 30 ng/ml) and the GrassrootsHealth cohort (N = 1,135), a prospective cohort (median 25(OH)D = 48 ng/ml). Cancer incidence over a multi-year period (median: 3.9 years) was compared according to 25(OH)D concentration. Kaplan-Meier plots were developed and the association between 25(OH)D and cancer risk was examined with multivariate Cox regression using multiple 25(OH)D measurements and spline functions. The study included all invasive cancers excluding skin cancer.ResultsAge-adjusted cancer incidence across the combined cohort (N = 2,304) was 840 cases per 100,000 person-years (1,020 per 100,000 person-years in the Lappe cohort and 722 per 100,000 person-years in the GrassrootsHealth cohort). Incidence was lower at higher concentrations of 25(OH)D. Women with 25(OH)D concentrations ≥40 ng/ml had a 67% lower risk of cancer than women with concentrations <20 ng/ml (HR = 0.33, 95% CI = 0.12–0.90).Conclusions25(OH)D concentrations ≥40 ng/ml were associated with substantial reduction in risk of all invasive cancers combined.