A method for the analysis of the oligomerization profile of the Huntington's disease-associated, aggregation-prone mutant huntingtin protein by isopycnic ultracentrifugation.

Conformational diseases, such as Alzheimer's, Parkinson's and Huntington's diseases as well as ataxias and fronto-temporal disorders, are part of common class of neurological disorders characterised by the aggregation and progressive accumulation of mutant proteins which display aberrant conformation. In particular, Huntington's disease (HD) is caused by mutations leading to an abnormal expansion in the polyglutamine (poly-Q) tract of the huntingtin protein (HTT), leading to the formation of inclusion bodies in neurons of affected patients. Furthermore, recent experimental evidence is challenging the conventional view of the disease by revealing the ability of mutant HTT to be transferred between cells by means of extracellular vesicles (EVs), allowing the mutant protein to seed oligomers involving both the mutant and wild type forms of the protein.

Lysosomal positioning diseases: beyond substrate storage.

Lysosomal storage diseases (LSDs) comprise a group of inherited monogenic disorders characterized by lysosomal dysfunctions due to undegraded substrate accumulation. They are caused by a deficiency in specific lysosomal hydrolases involved in cellular catabolism, or non-enzymatic proteins essential for normal lysosomal functions. In LSDs, the lack of degradation of the accumulated substrate and its lysosomal storage impairs lysosome functions resulting in the perturbation of cellular homeostasis and, in turn, the damage of multiple organ systems.

Protocol for labeling and fixation of intact lysosomes with esterified amino acid analogs to assess lysosomal expansion in living eukaryotic cells.

The lysosomal compartment is a key hub for cell metabolism, and morphological alterations have been described in several pathological conditions. Here, we describe the use of amino acid analogs modified by the presence of a methyl ester group that accumulates within lysosomes. This generates an intraluminal osmotic effect able to trigger a rapid and selective expansion of the lysosomal compartment within 2 h of treatment.

Early onset effects of single substrate accumulation recapitulate major features of LSD in patient-derived lysosomes.

Lysosome functions mainly rely on their ability to either degrade substrates or release them into the extracellular space. Lysosomal storage disorders (LSDs) are commonly characterized by a chronic lysosomal accumulation of different substrates, thereby causing lysosomal dysfunctions and secretion defects. However, the early effects of substrate accumulation on lysosomal homeostasis have not been analyzed so far.

An αB-Crystallin Peptide Rescues Compartmentalization and Trafficking Response to Cu Overload of ATP7B-H1069Q, the Most Frequent Cause of Wilson Disease in the Caucasian Population.

The H1069Q substitution is the most frequent mutation of the Cu transporter ATP7B that causes Wilson disease in the Caucasian population. ATP7B localizes to the Golgi complex in hepatocytes, but, in the presence of excessive Cu, it relocates to the endo-lysosomal compartment to excrete Cu via bile canaliculi. In contrast, ATP7B-H1069Q is strongly retained in the ER, does not reach the Golgi complex and fails to move to the endo-lysosomal compartment in the presence of excessive Cu, thus causing toxic Cu accumulation.

A disorder-to-order structural transition in the COOH-tail of Fz4 determines misfolding of the L501fsX533-Fz4 mutant.

Frizzled 4 belongs to the superfamily of G protein coupled receptors. The unstructured cytosolic tail of the receptor is essential for its activity. The mutation L501fsX533 in the fz4 gene results in a new COOH-tail of the receptor and causes a form of Familial exudative vitreoretinopathy.

Ligand of Numb proteins LNX1p80 and LNX2 interact with the human glycoprotein CD8α and promote its ubiquitylation and endocytosis.

E3 ubiquitin ligases give specificity to the ubiquitylation process by selectively binding substrates. Recently, their function has emerged as a crucial modulator of T-cell tolerance and immunity. However, substrates, partners and mechanism of action for most E3 ligases remain largely unknown.

Regulation of ERGIC-53 gene transcription in response to endoplasmic reticulum stress.

Accumulation of unfolded proteins within the endoplasmic reticulum (ER) activates the unfolded protein response, also known as the ER stress response. We previously demonstrated that ER stress induces transcription of the ER Golgi intermediate compartment protein ERGIC-53. To investigate the molecular events that regulate unfolded protein response-mediated induction of the gene, we have analyzed the transcriptional regulation of ERGIC-53.