SEPN1, an endoplasmic reticulum-localized selenoprotein linked to skeletal muscle pathology, counteracts hyperoxidation by means of redox-regulating SERCA2 pump activity.

Selenoprotein N (SEPN1) is a broadly expressed resident protein of the endoplasmic reticulum (ER) whose loss-of-function inexplicably leads to human muscle disease. We found that SEPN1 levels parallel those of endoplamic reticulum oxidoreductin 1 (ERO1), an ER protein thiol oxidase, and that SEPN1's redox activity defends the ER from ERO1-generated peroxides. Moreover, we have defined the redox-regulated interactome of SEPN1 and identified the ER calcium import SERCA2 pump as a redox-partner of SEPN1. SEPN1 enhances SERCA2 activity by reducing luminal cysteines that are hyperoxidized by ERO1-generated peroxides. Cells lacking SEPN1 are hypersensitive to ERO1 overexpression and conspicuously defective in ER calcium re-uptake. After being muscle-transduced with an adeno-associated virus driving ERO1α, SEPN1 knockout mice unmasks a myopathy that resembles the dense core disease due to human mutations in SEPN1, whereas the combined attenuation of ERO1α and SEPN1 enhances cell fitness. These observations reveal the involvement of SEPN1 in ER redox and calcium homeostasis and that an ERO1 inhibitor, restoring redox-dependent calcium homeostasis, may ameliorate the myopathy of SEPN1 deficiency.

The peculiar role of the A2V mutation in amyloid-ß (Aß) 1-42 molecular assembly.

We recently reported a novel Aß precursor protein mutation (A673V), corresponding to position 2 of Aß1-42 peptides (Aß1-42A2V), that caused an early onset AD-type dementia in a homozygous individual. The heterozygous relatives were not affected as an indication of autosomal recessive inheritance of this mutation. We investigated the folding kinetics of native unfolded Aß1-42A2V in comparison with the wild type sequence (Aß1-42WT) and the equimolar solution of both peptides (Aß1-42MIX) to characterize the oligomers that are produced in the early phases. We carried out the structural characterization of the three preparations using electron and atomic force microscopy, fluorescence emission, and x-ray diffraction and described the soluble oligomer formation kinetics by laser light scattering. The mutation promoted a peculiar pathway of oligomerization, forming a connected system similar to a polymer network with hydrophobic residues on the external surface. Aß1-42MIX generated assemblies very similar to those produced by Aß1-42WT, albeit with slower kinetics due to the difficulties of Aß1-42WT and Aß1-42A2V peptides in building up of stable intermolecular interaction.

Functional characterization of SbmA, a bacterial inner membrane transporter required for importing the antimicrobial peptide Bac7(1-35).

SbmA is an inner membrane protein of Gram-negative bacteria that is involved in the internalization of glycopeptides and prokaryotic and eukaryotic antimicrobial peptides, as well as of peptide nucleic acid (PNA) oligomers. The SbmA homolog BacA is required for the development of Sinorhizobium meliloti bacteroids within plant cells and favors chronic infections with Brucella abortus and Mycobacterium tuberculosis in mice. Here, we investigated functional features of SbmA/BacA using the proline-rich antimicrobial peptide Bac7(1-35) as a substrate. Circular dichroism and affinity chromatography studies were used to investigate the ability of SbmA to bind the peptide, and a whole-cell transport assay with fluorescently labeled peptide allowed the determination of transport kinetic parameters with a calculated Km value of 6.95 ± 0.89 µM peptide and a Vmax of 53.91 ± 3.17 nmol/min/mg SbmA. Use of a bacterial two-hybrid system coupled to SEC-MALLS (size exclusion chromatography coupled with multiangle laser light scattering) analyses established that SbmA is a homodimer in the membrane, and treatment of the cells with arsenate or ionophores indicated that the peptide transport mediated by SbmA is driven by the electrochemical gradient. Overall, these results shed light on the SbmA-mediated internalization of peptide substrates and suggest that the transport of an unknown substrate(s) represents the function of this protein.

Gramicidin A-based peptide vector for intracellular protein delivery.

The development of the peptide-based vectors for the intracellular delivery of biologically active macromolecules has opened new prospects of their application in research and therapy. Earlier the amphipathic cell-penetrating peptide (CPP) Pep-1 was reported to mediate cellular uptake of proteins without covalent binding to them. In this work we studied the ability of a series of membrane-active amphipathic peptides, based on the gramicidin A sequence, to transport a model protein across the eukaryotic cell membrane. Among them the positively charged Cys-containing peptide P10C demonstrated the most effective beta-galactosidase intracellular delivery. Besides, this peptide was shown to form noncovalent associates with beta-galactosidase as judged from electrophoresis and enzymatic activity assays. In addition, a series of new gramicidin analogues were prepared and the effect of N-terminus modification of gramicidin on the protein transduction efficiency was studied.

The A2V mutation as a new tool for hindering Aß aggregation: A neutron and x-ray diffraction study.

We have described a novel C-to-T mutation in the APP gene that corresponds to an alanine to valine substitution at position 673 in APP (A673V), or position 2 of the amyloid-ß (Aß) sequence. This mutation is associated with the early onset of AD-type dementia in homozygous individuals, whereas it has a protective effect in the heterozygous state. Correspondingly, we observed differences in the aggregation properties of the wild-type and mutated Aß peptides and their mixture. We have carried out neutron diffraction (ND) and x-ray diffraction (XRD) experiments on magnetically-oriented fibers of Aß1-28WT and its variant Aß1-28A2V. The orientation propensity was higher for Aß1-28A2V suggesting that it promotes the formation of fibrillar assemblies. The diffraction patterns by Aß1-28WT and Aß1-28A2V assemblies differed in shape and position of the equatorial reflections, suggesting that the two peptides adopt distinct lateral packing of the diffracting units. The diffraction patterns from a mixture of the two peptides differed from those of the single components, indicating the presence of structural interference during assembly and orientation. The lowest orientation propensity was observed for a mixture of Aß1-28WT and a short N-terminal fragment, Aß1-6A2V, which supports a role of Aß's N-terminal domain in amyloid fibril formation.

A new face for old antibiotics: tetracyclines in treatment of amyloidoses.

The use of tetracyclines has declined because of the appearance of resistant bacterial strains. However, the indications of nonantimicrobial activities of these drugs have considerably raised interest and triggered clinical trials for a number of different pathologies. About 10 years ago we first reported that tetracyclines inhibited the aggregation of prion protein fragments and Alzheimer's ß peptides, destabilizing their aggregates and promoting their degradation by proteases. On the basis of these observations, the antiamyloidogenic effects of tetracyclines on a variety of amyloidogenic proteins were studied and confirmed by independent research groups. In this review we comment on the data available on their antiamyloidogenic activity in preclinical and clinical studies. We also put forward that the beneficial effects of these drugs are a result of a peculiar pleiotropic action, comprising their interaction with oligomers and disruption of fibrils, as well as their antioxidant, anti-inflammatory, antiapoptotic, and matrix metalloproteinase inhibitory activities.

Epitope mapping of a PrP(Sc)-specific monoclonal antibody: identification of a novel C-terminally truncated prion fragment.

Monoclonal antibodies (mAbs) against prion proteins (PrPs) are indispensable in research and diagnosis of prion diseases, however the majority of these bind both the cellular (PrP(C)) and the disease-associated (PrP(Sc)) isoforms. According to the widely accepted protein-only hypothesis the two isoforms share the same sequence, but differ in their conformation. In the present study we set to determine the critical binding residues of our PrP(Sc)-specific mAbs with the view of discerning which residues play a key role in the conformational transition between PrP(C) and PrP(Sc). Focussing on the V5B2 mAb that provided differential labelling of prion-affected tissue from individuals positive for transmissible spongiform encephalopathies, we performed alanine scanning and phage-display epitope mapping to elucidate the antigenic determinants of this mAb and gain insight into its specificity on a molecular level. We observed that instead of discriminating between the two prion protein isoforms based on conformational differences, V5B2 binds a previously uncharacterized C-terminally truncated form of PrP(Sc) that ends with the residue Y226, which we named PrP226*. The addition of a single C-terminal amino-acid residue completely abolished V5B2 binding, while Western blots using recombinant full-length PrPs and PrPs terminating at Y226 confirmed that the V5B2 mAb discriminates between the two based on their difference in length.