The HSPB1-p62/SQSTM1 functional complex regulates the unconventional secretion and transcellular spreading of the HD-associated mutant huntingtin protein.

Conformational diseases, such as Alzheimer, Parkinson and Huntington diseases, are part of a common class of neurological disorders characterized by the aggregation and progressive accumulation of proteins bearing aberrant conformations. Huntington disease (HD) has autosomal dominant inheritance and is caused by mutations leading to an abnormal expansion in the polyglutamine (polyQ) tract of the huntingtin (HTT) protein, leading to the formation of HTT inclusion bodies in neurons of affected patients. Interestingly, recent experimental evidence is challenging the conventional view by which the disease pathogenesis is solely a consequence of the intracellular accumulation of mutant protein aggregates. These studies reveal that transcellular transfer of mutated huntingtin protein is able to seed oligomers involving even the wild-type (WT) forms of the protein. To date, there is still no successful strategy to treat HD. Here, we describe a novel functional role for the HSPB1-p62/SQSTM1 complex, which acts as a cargo loading platform, allowing the unconventional secretion of mutant HTT by extracellular vesicles. HSPB1 interacts preferentially with polyQ-expanded HTT compared with the WT protein and affects its aggregation. Furthermore, HSPB1 levels correlate with the rate of mutant HTT secretion, which is controlled by the activity of the PI3K/AKT/mTOR signalling pathway. Finally, we show that these HTT-containing vesicular structures are biologically active and able to be internalized by recipient cells, therefore providing an additional mechanism to explain the prion-like spreading properties of mutant HTT. These findings might also have implications for the turn-over of other disease-associated, aggregation-prone proteins.

Unconventional secretion of α-Crystallin B requires the Autophagic pathway and is controlled by phosphorylation of its serine 59 residue.

α-Crystallin B (CRYAB or HspB5) is a chaperone member of the small heat-shock protein family that prevents aggregation of many cytosolic client proteins by means of its ATP-independent holdase activity. Surprisingly, several reports show that CRYAB exerts a protective role also extracellularly, and it has been recently demonstrated that CRYAB is secreted from human retinal pigment epithelial cells by an unconventional secretion pathway that involves multi-vesicular bodies. Here we show that autophagy is crucial for this unconventional secretion pathway and that phosphorylation at serine 59 residue regulates CRYAB secretion by inhibiting its recruitment to the autophagosomes. In addition, we found that autophagosomes containing CRYAB are not able to fuse with lysosomes. Therefore, CRYAB is capable to highjack and divert autophagosomes toward the exocytic pathway, inhibiting their canonical route leading to the lysosomal compartment. Potential implications of these findings in the context of disease-associated mutant proteins turn-over are discussed.

Efficient microwave-assisted synthetic protocols and in silico behaviour prediction of per-substituted ß-cyclodextrins.

Selective per-substituted cyclodextrin design enables the carrier's physicochemical and binding properties to be tailored and can even modify some biological native structure effects. We herein report a number of highly efficient microwave-assisted synthetic protocols for the preparation of several amino, ureido and thioureido per-substituted ß-cyclodextrin derivatives. A rapid parallel synthetic approach has given a set of 14 different CD derivatives. Our strategy is supported by computational analyses which were used to estimate the physicochemical behaviour of per-substituted derivatives and to tailor suitable substituents.

Synthesis of water-soluble multidentate aminoalcohol ß-cyclodextrin derivatives via epoxide opening.

New highly soluble ß-aminoalcohol ß-cyclodextrin (ß-CD) derivatives have been synthesized via nucleophilic epoxide opening reactions with mono-6-amino mono-6-deoxy-permethyl-ß-CD and mono-6-amino mono-6-deoxy-ß-CD. The binding properties of the ß-CD were enhanced by linking aminoalcohol subunits which caused its solubility to improve markedly. The reaction conditions were optimised using microwave irradiation giving moderate-to-good yields with a series of epoxides. A regioselective epoxide opening reaction was observed in the reaction with styrene oxide while the stereoselectivity was strictly dependent on substrate structure.

Sequence and expression of the monkey homologue of the ER-golgi intermediate compartment lectin, ERGIC-53.

We obtained the cDNA sequence of the monkey homologue of the intermediate compartment protein ERGIC-53 by both cDNA library screening and RT-PCR amplification. The final sequence of 2422 nts of the monkey ERGIC-53 cDNA is 96.2% identical to the human ERGIC-53 cDNA and 87% and 67% identical to the rat and amphibian cDNA, respectively. The translated CV1 ERGIC-53 protein is 96.47% identical to the human ERGIC-53, 87% identical to the rat p58 and 66. 98% to the Xenopus laevis protein. Southern blot analysis of multiple genomic DNAs shows the presence of sequences similar to ERGIC-53 in different species. ERGIC-53 is expressed as a major transcript of about 5.5 kb in either monkey CV1 or in human CaCo2. A shorter transcript of 2.3 kb was detected in both cell lines and in mRNAs derived from human pancreas and placenta.

High resolution methylation analysis of the galectin-1 gene promoter region in expressing and nonexpressing tissues.

We conducted by bisulfite genomic sequencing a high resolution study of the methylation of the galectin-1 gene in expressing and nonexpressing tissues. We show that: (i) hypomethylation of galectin-1 promoter correlates with expression; (ii) differences in methylation occur in a small region, which include a CpG cluster; (iii) the density of methyl-CpGs rather than site-specific methylation distinguishes the nonexpressing from the expressing alleles; (iv) the modification profiles in nonexpressing tissues are highly heterogeneous; (v) a single CpG within 1300 bp is always methylated both in expressing and nonexpressing tissues; (vi) these features are conserved in rat and mouse.

A quantitative polymerase chain reaction (PCR) assay completely discriminates between Duchenne and Becker muscular dystrophy deletion carriers and normal females.

Duchenne/Becker muscular dystrophy (DMD/BMD) is a severe X-linked myopathy. In 65% of the patients, the mutations responsible for the disease are macrodeletions in the dystrophin-encoding gene that can be identified with multiplex polymerase chain reaction (PCR) technology. We developed a method for quantitative PCR analysis of deletion carriers involving the use of phosphorimager-based scanning of radioactive-labelled PCR products. We calculated the ratios between the areas of two peaks, one corresponding to the deleted segments to be analysed and the other taken as a reference. In carriers, these ratios (R value) were always about half those obtained in normal females. The final diagnostic result, the diagnostic index (DI), is the ratio of the R values between the propositus and a normal subject. We also assessed the variability of each step of the procedure and the overall variability of the DI value, thus obtaining cut-off values that completely discriminated BMD/DMD deletion carriers from normal females. We were also able to classify, as either 'carrier' or 'normal', several females whose status was not identified with linkage analysis.