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.     

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.     

Proteomics of rat biological fluids--the tenth anniversary update

In addition to a remarkable sexual dimorphism of serum and urine proteomes, the rat is exceptional for the wide difference between the serum patterns during an acute phase reaction vs baseline conditions. This feature allows monitoring with high sensitivity onset and progression of any pathological state that involves an inflammatory component as well as assessing the outcome of any therapeutic intervention. Reference maps have been defined for the proteomes of serum, urine, cerebrospinal fluid and bronchoalveolar lavage fluid. For both serum and urine most of the proteomic investigations have dealt with toxicological testing, for BALF with allergic or irritative reactions, whereas with CSF the main aim was the characterization of rat models of neurological disorders. When surveying more than ten years of literature on rat biological fluid proteomics, it is puzzling to see how seldom a consistent analytical plan has been set up for the comparative investigation on two or more types of sample, whether to fully characterize a disease model or to evaluate pharmacological/toxicological effects of a drug. It is also regrettable that in several cases only a negligible part of the results is discussed at length whereas most data are not even made known to the scientific community.     

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.     

Structural and dynamic roles of permanent water molecules in ligand molecular recognition by chicken liver bile acid binding protein

Chicken liver bile acid binding protein (cL-BABP) crystallizes with water molecules in its binding site. To obtain insights on the role of internal water, we performed two 100 ns molecular dynamics (MD) simulations in explicit solvent for cL-BABP, as apo form and as a complex with two molecules of cholic acid, and analyzed in detail the dynamics properties of all water molecules. The diffusion coefficients of the more persistent internal water molecules are significantly different from the bulk, but similar between the two protein forms. A different number of molecules and a different organization are observed for apo- and holo-cL-BABP. Most water molecules identified in the binding site of the apo-crystal diffuse to the bulk during the simulation. In contrast, almost all the internal waters of the holo-crystal maintain the same interactions with internal sidechains and ligands, which suggests they have a relevant role in protein-ligand molecular recognition. Only in the presence of these water molecules we were able to reproduce, by a classical molecular docking approach, the structure of the complex cL-BABP::cholic acid with a low ligand root mean square deviation (RMSD) with respect to its reference positioning. Literature data reported a conserved pattern of hydrogen bonds between a single water molecule and three amino acid residues of the binding site in a series of crystallized FABP. In cL-BABP, the interactions between this conserved water molecule and the three residues are present in the crystal of both apo- and holo-cL-BABP but are lost immediately after the start of molecular dynamics. Copyright (c) 2008 John Wiley & Sons, Ltd.     

Metal binding domains 3 and 4 of the Wilson disease protein: solution structure and interaction with the copper(I) chaperone HAH1

The Wilson disease protein or ATP7B is a P 1B-type ATPase involved in human copper homeostasis. The extended N-terminus of ATP7B protrudes into the cytosol and contains six Cu(I) binding domains. This report presents the NMR structure of the polypeptide consisting of soluble Cu(I) binding domains 3 and 4. The two domains exhibit ferredoxin-like folds, are linked by a flexible loop, and act independently of one another. Domains 3 and 4 tend to aggregate in a concentration-dependent manner involving nonspecific intermolecular interactions. Both domains can be loaded with Cu(I) when provided as an acetonitrile complex or by the chaperone HAH1. HAH1 forms a 70% complex with domain 4 that is in fast exchange with the free protein in solution. The ability of HAH1 to form a complex only with some domains of ATP7B is an interesting property of this class of proteins and may have a signaling role in the function of the ATPases.