Solution structure of human beta-parvalbumin and structural comparison with its paralog alpha-parvalbumin and with their rat orthologs

The aim of this research was to determine the structure of human beta-parvalbumin (109 amino acids) and to compare it with its paralog and ortholog proteins. The structure was determined in solution using multinuclear and multidimensional NMR methods and refined using substitution of the EF-hand Ca(2+) ion with a paramagnetic lanthanide. The resulting family of structures had a backbone rmsd of 0.50 A. Comparison with rat oncomodulin (X-ray, 1.3 A resolution) as well as with human (NMR, backbone rmsd of 0.49 A) and rat (X-ray, 2.0 A resolution) parvalbumins reveals small but reliable local differences, often but not always related to amino acid variability. The analysis of these structures has led us to propose an explanation for the different affinity for Ca(2+) between alpha- and beta-parvalbumins and between parvalbumins and calmodulins.     

The Atx1-Ccc2 complex is a metal-mediated protein-protein interaction

Cellular systems allow transition-metal ions to reach or leave the cell or intracellular locations through metal transfer between proteins. By coupling mutagenesis and advanced NMR experiments, we structurally characterized the adduct between the copper chaperone Atx1 and the first copper(I)-binding domain of the Ccc2 ATPase. Copper was required for the interaction. This study provides an understanding of metal-mediated protein-protein interactions in which the metal ion is essential for the weak, reversible interaction between the partners.     

Monomorphism of human cytochrome c

Cytochrome c (Cyt c) has key roles in both mitochondrial electron transfer and apoptosis onset and is therefore likely undergoing a strong selective pressure against amino acid variation. Nevertheless, a phylogenetically fast amino acid replacement rate in the Cyt c of species of the anthropoid primate lineage was recently reported. We therefore looked for the presence of nonsynonymous single nucleotide polymorphisms (nsSNPs) in the human Cyt c (HGNC approved gene symbol: CYCS), which, given its cellular constraints, could have important functional consequences, and found a large number of putative nsSNPs reported in the dbSNP database. We then subjected these putative SNPs to experimental validation by sequencing the Cyt c gene in a panel of 95 individuals assumed as a standard reference of the human population diversity. Surprisingly, none of the putative SNPs survived experimental validation. We conclude that non-rare allelic variants of the Cyt c protein are absent in the human populations analyzed in this study.     

Quantitative determination of callose in tree roots

The formation of callose in tree roots has been suggested as a physiological indicator of aluminum (Al) toxicity. Quantifying callose in the roots in forest soils, however, is hampered by the presence of autofluorescent materials in the roots that disturb the measurement of callose by fluorescence spectrophotometry. Tannins in the roots cause these measurement problems. Here we report on the measurement of callose in the root apices of European chestnut (Castanea sativa) seedlings collected in an acidified forest soil. The callose was quantified with a modified protocol which included three washing steps with polyvinylpolypyrrolidone (PVPP) before the callose was extracted. This procedure reduced the autofluorescence by about 50%. With the use of water or ethanol alone, callose could be measured in only about 15% of the root samples, whereas with the use of PVPP callose could be determined in 95% of the samples. This improved method could help to evaluate the effects of Al toxicity on tree roots grown in forest soils, where callose is detected as a physiological indicator.     

Human SOD1 before harboring the catalytic metal: solution structure of copper-depleted, disulfide-reduced form

SOD1 has to undergo several post-translational modifications before reaching its mature form. The protein requires insertion of zinc and copper atoms, followed by the formation of a conserved S-S bond between Cys-57 and Cys-146 (human numbering), which makes the protein fully active. In this report an NMR structural investigation of the reduced SH-SH form of thermostable E,Zn-as-SOD1 (E is empty; as is C6A, C111S) is reported, characterizing the protein just before the last step leading to the mature form. The structure is compared with that of the oxidized S-S form as well as with that of the yeast SOD1 complexed with its copper chaperone, CCS. Local conformational rearrangements upon disulfide bridge reduction are localized in the region near Cys-57 that is completely exposed to the solvent in the present structure, at variance with the oxidized forms. There is a local disorder around Cys-57 that may serve for protein-protein recognition and may possibly be involved in intermolecular S-S bonds in familial amyotrophic lateral sclerosis-related SOD1 mutants. The structure allows us to further discuss the copper loading mechanism in SOD1.     

Identification of N-acyl homoserine lactones produced by Gluconacetobacter diazotrophicus PAL5 cultured in complex and synthetic media

The endophytic diazotrophic Gluconacetobacter diazotrophicus PAL5 was originally isolated from sugarcane (Saccharum officinarum). The biological nitrogen fixation, phytohormones secretion, solubilization of mineral nutrients and phytopathogen antagonism allow its classification as a plant growth-promoting bacterium. The recent genomic sequence of PAL5 unveiled the presence of a quorum sensing (QS) system. QS are regulatory mechanisms that, through the production of signal molecules or autoinducers, permit a microbial population the regulation of the physiology in a coordinated manner. The most studied autoinducers in gram-negative bacteria are the N-acyl homoserine lactones (AHLs). The usage of biosensor strains evidenced the presence of AHL-like molecules in cultures of G. diazotrophicus PAL5 grown in complex and synthetic media. Analysis of AHLs performed by LC-APCI-MS permitted the identification of eight different signal molecules, including C6-, C8-, C10-, C12- and C14-HSL. Mass spectra confirmed that this diazotrophic strain also synthesizes autoinducers with carbonyl substitutions in the acyl chain. No differences in the profile of AHLs could be determined under both culture conditions. However, although the level of short-chain AHLs was not affected, a decrease of 30% in the production of long-chain AHLs could be measured in synthetic medium.     

miR-24 triggers epidermal differentiation by controlling actin adhesion and cell migration

During keratinocyte differentiation and stratification, cells undergo extensive remodeling of their actin cytoskeleton, which is important to control cell mobility and to coordinate and stabilize adhesive structures necessary for functional epithelia. Limited knowledge exists on how the actin cytoskeleton is remodeled in epithelial stratification and whether cell shape is a key determinant to trigger terminal differentiation. In this paper, using human keratinocytes and mouse epidermis as models, we implicate miR-24 in actin adhesion dynamics and demonstrate that miR-24 directly controls actin cable formation and cell mobility. miR-24 overexpression in proliferating cells was sufficient to trigger keratinocyte differentiation both in vitro and in vivo and directly repressed cytoskeletal modulators (PAK4, Tks5, and ArhGAP19). Silencing of these targets recapitulated the effects of miR-24 overexpression. Our results uncover a new regulatory pathway involving a differentiation-promoting microribonucleic acid that regulates actin adhesion dynamics in human and mouse epidermis.     

On the use of ultracentrifugal devices for sedimented solute NMR

We have recently proposed sedimented solute NMR (SedNMR) as a solid-state method to access biomolecules without the need of crystallization or other sample manipulation. The drawback of SedNMR is that samples are intrinsically diluted and this is detrimental for the signal intensity. Ultracentrifugal devices can be used to increase the amount of sample inside the rotor, overcoming the intrinsic sensitivity limitation of the method. We designed two different devices and we here report the directions for using such devices and the relevant equations for determining the parameters for sedimentation.     

Cedar Virus: A Novel Henipavirus Isolated from Australian Bats

The genus Henipavirus in the family Paramyxoviridae contains two viruses, Hendra virus (HeV) and Nipah virus (NiV) for which pteropid bats act as the main natural reservoir. Each virus also causes serious and commonly lethal infection of people as well as various species of domestic animals, however little is known about the associated mechanisms of pathogenesis. Here, we report the isolation and characterization of a new paramyxovirus from pteropid bats, Cedar virus (CedPV), which shares significant features with the known henipaviruses. The genome size (18,162 nt) and organization of CedPV is very similar to that of HeV and NiV; its nucleocapsid protein displays antigenic cross-reactivity with henipaviruses; and it uses the same receptor molecule (ephrin- B2) for entry during infection. Preliminary challenge studies with CedPV in ferrets and guinea pigs, both susceptible to infection and disease with known henipaviruses, confirmed virus replication and production of neutralizing antibodies although clinical disease was not observed. In this context, it is interesting to note that the major genetic difference between CedPV and HeV or NiV lies within the coding strategy of the P gene, which is known to play an important role in evading the host innate immune system. Unlike HeV, NiV, and almost all known paramyxoviruses, the CedPV P gene lacks both RNA editing and also the coding capacity for the highly conserved V protein. Preliminary study indicated that CedPV infection of human cells induces a more robust IFN-β response than HeV.