Characterization of oligomeric intermediates in alpha-synuclein fibrillation: FRET studies of Y125W/Y133F/Y136F alpha-synuclein

The aggregation of alpha-synuclein is believed to be a critical step in the etiology of Parkinson's disease. A variety of biophysical techniques were used to investigate the aggregation and fibrillation of alpha-synuclein in which one of the four intrinsic Tyr residues was replaced by Trp, and two others by Phe, in order to permit fluorescence resonance energy transfer (FRET) between residues 39 (Tyr) and 125 (Trp). The mutant Y125W/Y133F/Y136F alpha-synuclein (one Tyr, one Trp) showed fibrillation kinetics similar to that of the wild-type, as did the Y125F/Y133F/Y136F (one Tyr, no Trp) and Y39F/Y125W/Y133F/Y136F (no Tyr, one Trp) mutants. Time-dependent changes in FRET, Fourier transform infrared, Trp fluorescence, dynamic light-scattering and other probes, indicate the existence of a transient oligomer, whose population reaches a maximum at the end of the lag time. This oligomer, in which the alpha-synuclein is in a partially folded conformation, is subsequently converted into fibrils, and has physical properties that are distinct from those of the monomer and fibrils. In addition, another population of soluble oligomers was observed to coexist with fibrils at completion of the reaction. The average distance between Tyr39 and Trp125 decreases from 24.9A in the monomer to 21.9A in the early oligomer and 18.8A in the late oligomer. Trp125 remains solvent-exposed in both the oligomers and fibrils, indicating that the C-terminal domain is not part of the fibril core. No FRET was observed in the fibrils, due to quenching of Tyr39 fluorescence in the fibril core. Thus, aggregation of alpha-synuclein involves multiple oligomeric intermediates and competing pathways.     

Mutations at the Arabidopsis CHM locus promote rearrangements of the mitochondrial genome.

Nuclear recessive mutations at the chloroplast mutator (CHM) locus of Arabidopsis produce a variegated phenotype that is inherited in a non-Mendelian fashion. Molecular analysis of the cytoplasmic genomes of variegated plants from two independent chm mutant lines, using specific chloroplast and mitochondrial probes, showed that the chm mutations reproducibly induce the appearance of specific new restriction fragments in the mitochondrial genome. The presence of these restriction fragments cosegregated with the variegated phenotype in the progeny of crosses between mutant and wild-type plants. Sequence analysis of one of the new restriction fragments found in the variegated plants suggested that it was the product of a rearrangement event involving regions of the mitochondrial genome. Thus, it appears that the CHM locus may encode a protein involved in the control of specific mitochondrial DNA reorganization events.     

Chilling-sensitive mutants of arabidopsis

Twenty-one mutants ofArabidopsis thaliana were isolated that developed chlorosis or necrosis upon incubation at low temperature (10°C to 15°C). Crosses among mutants in different phenotypic classes showed that mutants in three of four classes were found in a small number of loci. This article is reproduced fromWeeds World, vol. 1. For electronic access toWeeds World, see PMBR 12(4):302–303.     

Microarray data quality analysis: lessons from the AFGC project

Genome-wide expression profiling with DNA microarrays has and will provide a great deal of data to the plant scientific community. However, reliability concerns have required the development data quality tests for common systematic biases. Fortunately, most large-scale systematic biases are detectable and some are correctable by normalization. Technical replication experiments and statistical surveys indicate that these biases vary widely in severity and appearance. As a result, no single normalization or correction method currently available is able to address all the issues. However, careful sequence selection, array design, experimental design and experimental annotation can substantially improve the quality and biological of microarray data. In this review, we discuss these issues with reference to examples from the Arabidopsis Functional Genomics Consortium (AFGC) microarray project.     

Spatiotemporal dynamics of adenovirus membrane rupture and endosomal escape

A key step in adenovirus cell entry is viral penetration of cellular membranes to gain access to the cytoplasm and deliver the genome to the nucleus. Yet little is known about this important event in the adenoviral life cycle. Using the cytosolic protein galectin-3 (gal3) as a marker of membrane rupture with both live- and fixed-cell imaging, we demonstrate that in the majority of instances, exposure of pVI and recruitment of gal3 to ruptured membranes occur early at or near the cell surface and occur minimally in EEA-1-positive (EEA-1(+)) early endosomes or LAMP-1(+) late endosomes/lysosomes. Live-cell imaging of Ad5 egress from gal3(+) endosomes occurs most frequently from perinuclear locations. While the Ad5 capsid is observed escaping from gal3(+) endosomes, pVI appears to remain associated with the gal3(+) ruptured endosomes. Thus, Ad5 membrane rupture and endosomal escape appear to be both spatially and temporally distinct events.     

Role of Cell-Type-Specific Endoplasmic Reticulum-Associated Degradation in Polyomavirus Trafficking

BK polyomavirus (BKPyV) is a widespread human pathogen that establishes a lifelong persistent infection and can cause severe disease in immunosuppressed patients. BKPyV is a nonenveloped DNA virus that must traffic through the endoplasmic reticulum (ER) for productive infection to occur; however, it is unknown how BKPyV exits the ER before nuclear entry. In this study, we elucidated the role of the ER-associated degradation (ERAD) pathway during BKPyV intracellular trafficking in renal proximal tubule epithelial (RPTE) cells, a natural host cell. Using proteasome and ERAD inhibitors, we showed that ERAD is required for productive entry. Altered trafficking and accumulation of uncoated viral intermediates were detected by fluorescence in situ hybridization and indirect immunofluorescence in the presence of an inhibitor. Additionally, we detected a change in localization of partially uncoated virus within the ER during proteasome inhibition, from a BiP-rich area to a calnexin-rich subregion, indicating that BKPyV accumulated in an ER subcompartment. Furthermore, inhibiting ERAD did not prevent entry of capsid protein VP1 into the cytosol from the ER. By comparing the cytosolic entry of the related polyomavirus simian virus 40 (SV40), we found that dependence on the ERAD pathway for cytosolic entry varied between the polyomaviruses and between different cell types, namely, immortalized CV-1 cells and primary RPTE cells.     

Secondary nucleating sequences affect kinetics and thermodynamics of tau aggregation

Tau protein was scanned for highly amyloidogenic sequences in amphiphilic motifs (X)(n)Z, Z(X)(n)Z (n ≥ 2), or (XZ)(n) (n ≥ 2), where X is a hydrophobic residue and Z is a charged or polar residue. N-Acetyl peptides homologous to these sequences were used to study aggregation. Transmission electron microscopy (TEM) showed seven peptides, in addition to well-known primary nucleating sequences Ac(275)VQIINK (AcPHF6*) and Ac(306)VQIVYK (AcPHF6), formed fibers, tubes, ribbons, or rolled sheets. Of the peptides shown by TEM to form amyloid, Ac(10)VME, AcPHF6*, Ac(375)KLTFR, and Ac(393)VYK were found to enhance the fraction of β-structure of AcPHF6 formed at equilibrium, and Ac(375)KLTFR was found to inhibit AcPHF6 and AcPHF6* aggregation kinetics in a dose-dependent manner, consistent with its participation in a hybrid steric zipper model. Single site mutants were generated which transformed predicted amyloidogenic sequences in tau into non-amyloidogenic ones. A M11K mutant had fewer filaments and showed a decrease in aggregation kinetics and an increased lag time compared to wild-type tau, while a F378K mutant showed significantly more filaments. Our results infer that sequences throughout tau, in addition to PHF6 and PHF6*, can seed amyloid formation or affect aggregation kinetics or thermodynamics.