SUMOylation of the brain-predominant Ataxin-3 isoform modulates its interaction with p97.

BACKGROUND: Machado-Joseph Disease (MJD), a form of dominantly inherited ataxia belonging to the group of polyQ expansion neurodegenerative disorders, occurs when a threshold value for the number of glutamines in Ataxin-3 (Atx3) polyglutamine region is exceeded. As a result of its modular multidomain architecture, Atx3 is known to engage in multiple macromolecular interactions, which might be unbalanced when the polyQ tract is expanded, culminating in the aggregation and formation of intracellular inclusions, a unifying fingerprint of this group of neurodegenerative disorders. Since aggregation is specific to certain brain regions, localization-dependent posttranslational modifications that differentially affect Atx3 might also contribute for MJD. METHODS: We combined in vitro and cellular approaches to address SUMOylation in the brain-predominant Atx3 isoform and assessed the impact of this posttranslational modification on Atx3 self-assembly and interaction with its native partner, p97. RESULTS: We demonstrate that Atx3 is SUMOylated at K356 both in vitro and in cells, which contributes for decreased formation of amyloid fibrils and for increased affinity towards p97. CONCLUSIONS AND GENERAL SIGNIFICANCE: These findings highlight the role of SUMOylation as a regulator of Atx3 function, with implications on Atx3 protein interaction network and self-assembly, with potential impact for further understanding the molecular mechanisms underlying MJD pathogenesis.

Genotypic and phenotypic characteristics of Dutch patients with early onset Parkinson's disease.

Early onset Parkinson's disease (EOPD) has been associated with mutations in the Parkin, DJ-1, PINK1, LRRK2, and SNCA genes. The aim of this study is to assess the contribution of these genes in a Dutch EOPD cohort and the phenotypic characteristics of the mutation carriers. A total of 187 unrelated Dutch EOPD patients (age at onset < or = 50 years) were phenotyped and screened for mutations in all exons of Parkin, DJ-1, and PINK1 by direct sequencing and gene dosage analysis. Additionally, analysis of the A30P mutation and exon dosage of SNCA and sequencing of exons 19,31,35,38,41, and 48 of LRRK2 was performed. Pathogenic variations could explain disease in 4% (7 of 187) of the patients including five patients carrying homozygous or compound heterozygous mutations in Parkin, one with a novel homozygous deletion in DJ-1 (P158Del) and one with a heterozygous mutation in LRRK2 (T2356I). We found seven novel mutations. The phenotypic characteristics of mutation carriers varied widely, comparable to the variability seen in sporadic EOPD. Parkin is the most frequently mutated gene in this EOPD cohort, followed by DJ-1, PINK1 and LRRK2. The low overall mutation frequency indicates that the extrapolation of mutation frequencies from other populations should be applied with caution.

Molecular analysis of iron overload in beta2-microglobulin-deficient mice.

Beta2-microglobulin knockout (beta2m-/-) mice represent an instructive model of spontaneous iron overload resembling genetic hemochromatosis. The mechanism of iron accumulation in this mouse model may be more complex than involving the MHC class I-like protein HFE. We report that beta2m-deficient mice, like Hfe-/- mice, lack the adaptive hepatic hepcidin mRNA increase to iron overload. The inverse correlation of hepatic iron levels and hepcidin mRNA expression in six beta2m-/- mice underlines the importance of hepcidin in regulating body iron stores. In contrast to Hfe-/- mice, beta2m-deficient mice display increased expression of the duodenal iron transporters DMT1 and ferroportin 1. This result implicates a broader role of beta2m in mammalian iron metabolism, suggesting that (an) additional beta2m-interacting protein(s) could be involved in controlling iron homeostasis, and highlighting the emerging connection of iron metabolism with the immune system.

The DJ-1L166P mutant protein associated with early onset Parkinson's disease is unstable and forms higher-order protein complexes.

Parkinson's disease (PD) is a common neurodegenerative disorder that involves the selective degeneration of midbrain dopaminergic neurons. Recently DJ-1 mutations have been linked to autosomal-recessive early-onset Parkinsonism in two European families. By using gel filtration assays under physiological conditions we demonstrate that DJ-1 protein forms a dimeric structure. Conversely, the DJ-1L166P mutant protein shows a different elution profile as compared with DJ-1WT both in overexpression cellular systems or in lymphoblasts cells, suggesting that it might form higher order protein structures. Furthermore we observed that the level of DJ-1L166P mutant protein in the patient's lymphoblasts was very low as compared with the wild-type protein. We excluded a potential transcriptional impairment by performing quantitative RT-PCR on the patient's material. Pulse-chase experiments in transfected COS-1 cells and cycloheximide treatment in control and patient lymphoblasts indicated that the mutant protein was rapidly degraded. This rapid turnover and the structural changes of DJ-1L166P mutant protein might be crucial in the disease pathogenesis.

Increased hepatic iron in mice lacking classical MHC class I molecules.

Iron accumulation in the liver in hereditary hemochromatosis (HH) has been shown to be highly variable. Some studies point to the importance of major histocompatibility complex (MHC) class I (MHC-I) and CD8(+) cells as modifiers of iron overload. In this report, using mice knockout for H2K(b-/-) and H2D(b-/-) genes, it is demonstrated that lack of classical MHC-I molecules results in a spontaneous increase of nonheme iron content in the liver (mainly located in the hepatocytes) when compared to wild-type mice. In CD8(-/-) and Rag2(-/-) mice, no spontaneous hepatic iron accumulation was observed. These results demonstrate for the first time that classical MHC-I molecules could be involved in the regulation of iron metabolism and contribute to the established genotype/phenotype discrepancies seen in HH.

Combined effects of temperature and medium composition on exopolysaccharide production by Lactobacillus rhamnosus RW-9595M in a whey permeate based medium.

The effects of temperature (22-42 degrees C), whey permeate concentration (WP, 1.6-8.4%), and supplementation level with yeast nitrogen base (YNB, 0-2.0%) on exopolysaccharide (EPS) production was studied during 20 pH-controlled (pH = 6.0) batch cultures with Lactobacillus rhamnosus RW-9595M, using a central composite design (CCD). The EPS production was measured using both the conventional method based on ethanol precipitation of EPS and a new ultrafiltration (UF) method. EPS production was not growth-associated for high temperatures (32-42 degrees C) and WP concentrations (7.0-8.4%). In contrast, at suboptimal temperature (22-26 degrees C), EPS production was growth-associated. Maximal EPS production measured with the UF method was approximately 2-fold higher than those measured with the conventional method and varied from 125 to 477 mg/L. This parameter was significantly influenced by WP and YNBWP interaction, whereas ANOVA for maximal EPS production measured by the conventional method did not show significant factor effects. EPS volumetric productivities varied from 3.0 to 16.4 mg EPS/L small middle doth. YNB supplementation did not promote cell growth but did increase EPS production at high WP concentrations. Our data indicate the potential of L. rhamnosus RW-9595M for producing EPS in a supplemented WP medium and suggest that this production could be further increased by the addition of a growth-limiting nutrient in the medium.

Quantification of exopolysaccharide, lactic acid, and lactose concentrations in culture broth by near-infrared spectroscopy.

Near-infrared spectroscopy (NIRS) was used for the simultaneous prediction of exopolysaccharide (EPS; 0-3 g/L) and lactic acid (0-59 g/L) productions as well as lactose (0-68 g/L) concentration in supernatant samples from pH-controlled batch cultures of Lactobacillus rhamnosus RW-9595M in supplemented whey permeate medium. To develop calibration equations, the correlation between the second derivative of 164 NIRS transmittance spectra and concentration data obtained with reference methods was calculated at the wavelength between 1653-1770 and 2041-2353 nm, using a partial least-squares method (PLS). The lactic acid and lactose concentrations were measured by HPLC, and the EPS concentration was estimated by a new ultrafiltration method. The PLS correlation coefficient (R(2)) and the standard error of cross-validation for the calibrations were 91% and 0.26 g/L for EPS, 99% and 2.54 g/L for lactic acid, and 98% and 3.32 g/L for lactose, respectively. The calibration equations were validated with 45 randomly selected culture samples from 6 cultures that were not used for calibration. A high agreement between data of the reference methods and those of NIRS was observed, with correlation coefficients and standard errors of prediction of 99% and 1.64 g/L for lactic acid, 99% and 4.5 g/L for lactose, and 91% and 0.32 g/L for EPS. The results suggest that NIRS could be a useful method for rapid monitoring and control of EPS lactic fermentations.