Steady-State Kinetics of α-Synuclein Ferrireductase Activity Identifies the Catalytically Competent Species.

α-Synuclein (α-syn) is a cytosolic protein known for its association with neurodegenerative diseases, including Parkinson's disease and other synucleinopathies. The potential cellular function of α-synuclein may be of consequence for understanding the pathogenesis of such diseases. Previous work has suggested that α-synuclein can catalyze the reduction of iron as a ferrireductase. We performed a detailed analysis of the steady-state kinetics of recombinant α-syn ferrireductase activity and for disease-associated variants. Our study illustrates that the ferrireductase activity we observed is clearly commensurate with bona fide enzyme activity and suggests a mechanistic rationale for the activity and the relationship to cellular regulation of the pool of Fe(III) and Fe(II). Using cell-based studies, we examined the functionally active conformation and found that the major catalytically active form is a putative membrane-associated tetramer. Using an artificial membrane environment with recombinant protein, we demonstrate that secondary structure folding of α-synuclein is insufficient to allow enzyme activity and the absolute specificity of the tertiary/quaternary structure is the primary requirement. Finally, we explored the steady-state kinetics of a range of disease α-synuclein variants and found that variants involved in neurodegenerative disease exhibited major changes in their enzymatic activity. We discuss these data in the context of a potential disease-associated mechanism for aberrant α-synuclein ferrireductase activity.

Truncated RASSF7 promotes centrosomal defects and cell death.

RASSF7 protein localises to the centrosome and plays a key role in mitosis. Its expression is also increased in a range of tumour types. However, little is known about the molecular basis of RASSF7's function and it is not clear if it acts as an oncogene in the cancers where its levels are elevated. Here, we carry out the first analysis of the domains of rassf7, focusing on which of them are responsible for its localisation to the centrosome. Constructs were generated to allow the expression of a series of truncated versions of rassf7 and the level of centrosomal localisation shown by each protein quantified. This analysis was carried out in Xenopus embryos which are a tractable system where rassf7 localisation can easily be studied. Our data shows that the coiled-coil domain of rassf7 is required and sufficient to direct its centrosomal localisation. The RA domain did not appear to have a role in mediating localisation. Surprisingly, removal of the extreme C-terminus of the protein caused rassf7 to accumulate at the centrosome and drive centrosome defects, including accumulation of the centrosomal protein γ-tubulin and an amplification of the number of γ-tubulin foci. These effects required the centrosomal localisation mediated by the coiled-coil domain. Later in development cells expressing this truncated rassf7 protein underwent cell death. Finally, analysis of a database of tumour sequences identified a mutation in RASSF7 which would cause a similar C-terminal truncation of the protein. Based on our data this truncated protein might drive centrosomal defects and we propose the hypothesis that truncated RASSF7 could act as an oncogene in a small subset of tumours where it is mutated in this way.

GLUT4 traffic through an ESCRT-III-dependent sorting compartment in adipocytes.

In insulin target tissues, GLUT4 is known to traffic through multiple compartments that may involve ubiquitin- and/or SUMO-dependent targeting. During these trafficking steps, GLUT4 is sorted into a storage reservoir compartment that is acutely released by insulin signalling processes that are downstream of PI 3-kinase associated changes in inositol phospholipids. As ESCRT components have recently been found to influence cellular sorting processes that are related to changes in both ubiquitination and inositol phospholipids, we have examined whether GLUT4 traffic is routed through ESCRT dependent sorting steps. Introduction of the dominant negative inhibitory constructs of the ESCRT-III components CHMP3 (CHMP3(1-179)) and Vps4 (GFP-Vps4(E235Q)) into rat adipocytes leads to the accumulation of GLUT4 in large, coalesced and extended vesicles structures that co-localise with the inhibitory constructs over large parts of the extended structure. A new swollen hybrid and extensively ubiquitinated compartment is produced in which GLUT4 co-localises more extensively with the endosomal markers including EEA1 and transferrin receptors but also with the TGN marker syntaxin6. These perturbations are associated with failure of insulin action on GLUT4 traffic to the cell surface and suggest impairment in an ESCRT-dependent sorting step used for GLUT4 traffic to its specialised reservoir compartment.