Phosphorylation pattern of the NDUFS4 subunit of complex I of the mammalian respiratory chain

The NDUFS4 subunit of complex I of the mammalian respiratory chain has a fully conserved carboxy-terminus with a canonical RVSTK phosphorylation site. Immunochemical analysis with specific antibodies shows that the serine in this site of the protein is natively present in complex I in both the phosphorylated and non-phosphorylated state. Two-dimensional IEF/SDS–PAGE electrophoresis, ³²P labelling and immunodetection show that “in vitro” PKA phosphorylates the serine in the C-terminus of the NDUFS4 subunit in isolated bovine complex I. ³²P labelling and TLC phosphoaminoacid mapping show that PKA phosphorylates serine and threonine residues in the purified heterologous human NDUFS4 protein.     

Surface adsorption of protein corona controls the cell internalization mechanism of DC-Chol-DOPE/DNA lipoplexes in serum

Serum has often been reported as a barrier to efficient lipid-mediated transfection. Here we found that the transfection efficiency of DC-Chol-DOPE/DNA lipoplexes increases in serum. To provide insight into the mechanism of lipoplex-serum interaction, several state-of-the-art methodologies have been applied. The nanostructure of DC-Chol-DOPE/DNA lipoplexes was found to be serum-resistant as revealed by high resolution synchrotron small angle X-ray scattering, while dynamic light scattering measurements showed a marked size increase of complexes. The structural stability of DC-Chol-DOPE/DNA lipoplexes was confirmed by electrophoresis on agarose gel demonstrating that plasmid DNA remained well protected by lipids. Proteomics experiments showed that serum proteins competed for the cationic surface of lipid membranes leading to the formation of a rich a ‘protein corona’. Combining structural results with proteomics findings, we suggest that such a protein corona can promote large aggregation of intact lipoplexes. According to a recently proposed size-dependent mechanism of lipoplex entry within cells, protein corona-induced formation of large aggregates most likely results in a switch from a clathrin-dependent to caveolae-mediated entry pathway into the cells which is likely to be responsible for the observed transfection efficiency boost. As a consequence, we suggest that surface adsorption of protein corona can have a high biological impact on serum-resistant cationic formulations for in vitro and in vivo lipid-mediated gene delivery applications.     

Proteomic profiling of cerebrospinal fluid in Creutzfeldt-Jakob disease

Creutzfeldt-Jakob disease (CJD) is a rare fatal neurodegenerative disease belonging to the group of transmissible spongiform encephalopathies or prion diseases. The agent responsible for the disease is the prion protein in an altered conformational form. Although there have been countless studies performed on the prion protein, the mechanisms that induce the structural change of the normal protein, and the harmful action the altered protein has on nervous cells, are still not fully understood. Furthermore, the final diagnosis for CJD can only occur with a postmortem histopathological analysis of the brain; the antemortem diagnosis is only possible for some specific CJD forms. Finally, there is no current treatment able to stop or delay the progression of the disease. Studies directed at resolving these issues are, therefore, extremely relevant. The proteomic approach is a very good strategy to be applied in such contexts because it allows easy identification of proteins and peptides possibly involved in the disease processes. In this article, the existing data regarding prion infection, biomarkers for CJD diagnosis and the use of several modern proteomic technologies for the identification of new cerebrospinal fluid polypeptides involved in CJD are reviewed.     

Growth function of self-complementary circular codes.

In several papers Arquès and Michel studied the maximal circular codes consisting of words of length 3 (or trinucleotides) on the genetic alphabet {A, C, G, T}. We present here some additional information on these codes. In particular, we study the growth function of the self-complementary circular codes and we prove that among them exactly 528 are maximal.     

Type 1 IFN mediates cross-talk between innate and adaptive immunity that abrogates transplantation tolerance

TLR activation of innate immunity prevents the induction of transplantation tolerance and shortens skin allograft survival in mice treated with costimulation blockade. The mechanism by which TLR signaling mediates this effect has not been clear. We now report that administration of the TLR agonists LPS (TLR4) or polyinosinic:polycytidylic acid (TLR3) to mice treated with costimulation blockade prevents alloreactive CD8(+) T cell deletion, primes alloreactive CTLs, and shortens allograft survival. The TLR4- and MyD88-dependent pathways are required for LPS to shorten allograft survival, whereas polyinosinic:polycytidylic acid mediates its effects through a TLR3-independent pathway. These effects are all mediated by signaling through the type 1 IFN (IFN-alphabeta) receptor. Administration of IFN-beta recapitulates the detrimental effects of TLR agonists on transplantation tolerance. We conclude that the type 1 IFN generated as part of an innate immune response to TLR activation can in turn activate adaptive immune responses that abrogate transplantation tolerance. Blocking of type 1 IFN-dependent pathways in patients may improve allograft survival in the presence of exogenous TLR ligands.     

Low-resolution structures of thyroid hormone receptor dimers and tetramers in solution

High-resolution X-ray structures of thyroid hormone (TH) receptor (TR) DNA and ligand binding domains (DBD and LBD) have yielded significant insights into TR action. Nevertheless, the TR DBD and LBD act in concert to mediate TH effects upon gene expression, and TRs form multiple oligomers; however, structures of full-length TRs or DBD-LBD constructs that would clarify these influences are not available. Here, we report low-resolution X-ray structures of the TRbeta DBD-LBD construct in solution which define the shape of dimers and tetramers and likely positions of the DBDs and LBDs. The holo TRbeta DBD-LBD construct forms a homodimer with LBD-DBD pairs in close contact and DBDs protruding from the base in the same direction. The DBDs are connected to the LBDs by crossed extended D domains. The apo hTRbeta DBD-LBD construct forms tetramers that resemble bulged cylinders with pairs of LBD dimers in a head-to-head arrangement with DBD pairs packed tightly against the LBD core. Overall, there are similarities with our previous low-resolution structures of retinoid X receptors, but TRs exhibit two unique features. First, TR DBDs are closely juxtaposed in the dimer and tetramer forms. Second, TR DBDs are closely packed against LBDs in the tetramer, but not the dimer. These findings suggest that TRs may be able to engage in hitherto unknown interdomain interactions and that the D domain must rearrange in different oligomeric forms. Finally, the data corroborate our suggestion that apo TRs form tetramers in solution which dissociate into dimers upon hormone binding.     

SERCA2a, phospholamban, sarcolipin, and ryanodine receptors gene expression in children with congenital heart defects

In animal models of conotruncal heart defects, an abnormal calcium sensitivity of the contractile apparatus and a depressed L-type calcium current have been described. Sarcoplasmic reticulum (SR) Ca(2+) ATPase (SERCA) is a membrane protein that catalyzes the ATP-dependent transport of Ca(2+) from the cytosol to the SR. The activity of SERCA is inhibited by phospholamban (PLN) and sarcolipin (SLN), and all these proteins participate in maintaining the normal intracellular calcium handling. Ryanodine receptors (RyRs) are the major SR calcium-release channels required for excitation-contraction coupling in skeletal and cardiac muscle. Our objective was to evaluate SERCA2a (i.e., the SERCA cardiac isoform), PLN, SLN, and RyR2 (i.e., the RyR isoform enriched in the heart) gene expression in myocardial tissue of patients affected by tetralogy of Fallot (TOF), a conotruncal heart defect. The gene expression of target genes was assessed semiquantitatively by RT-PCR using the calsequestrin (CASQ, a housekeeping gene) RNA as internal standard in the atrial myocardium of 23 pediatric patients undergoing surgical correction of TOF, in 10 age-matched patients with ventricular septal defect (VSD) and in 13 age-matched children with atrial septal defect (ASD). We observed a significantly lower expression of PLN and SLN in TOF patients, while there was no difference between the expression of SERCA2a and RyR2 in TOF and VSD. These data suggest a complex mechanism aimed to enhance the intracellular Ca(2+) reserve in children affected by tetralogy of Fallot.