Karen A. Lewis

A Precipitating Role for Truncated α-Synuclein and the Proteasome in α-Synuclein Aggregation IMPLICATIONS FOR PATHOGENESIS OF PARKINSON DISEASE

Parkinson disease and other α-synucleinopathies are characterized by the deposition of intraneuronal α-synuclein (αSyn) inclusions. A significant fraction (about 15%) of αSyn in these pathological structures are truncated forms that have a much higher propensity than the full-length αSyn to form aggregates in vitro. However, little is known about the role of truncated αSyn species in pathogenesis or the means by which they are generated. Here, we have provided an in vitro mechanistic study demonstrating that truncated αSyns induce rapid aggregation of full-length protein at substoichiometric ratios. Co-overexpression of truncated αSyn with full-length protein increases cell vulnerability to oxidative stress in dopaminergic SH-SY5Y cells. These results suggest a precipitating role for truncated αSyn in the pathogenesis of diseases involving αSyn aggregation. In this regard, the A53T mutation found in some cases of familial Parkinson disease exacerbates the accumulation of insoluble αSyns that correlates with the onset of pathology in transgenic mice expressing human αSyn-A53T mutant. The caspase-like activity of the 20 S proteasome produces truncated fragments similar to those found in patients and animal models from degradation of unstructured αSyn. We propose a model in which incomplete degradation of αSyn, especially under overloaded proteasome capacity, produces highly amyloidogenic fragments that rapidly induce the aggregation of full-length protein. These aggregates in turn reduce proteasome activity, leading to further accumulation of fragmented and full-length αSyns, creating a vicious cycle of cytotoxicity. This model has parallels in other neurodegenerative diseases, such as Huntington disease, where coaggregation of poly(Q) fragments with full-length protein has been observed.

Anatomy of Escherichia coli σ70 promoters

Information theory was used to build a promoter model that accounts for the −10, the −35 and the uncertainty of the gap between them on a common scale. Helical face assignment indicated that base −7, rather than −11, of the −10 may be flipping to initiate transcription. We found that the sequence conservation of σ70 binding sites is 6.5 ± 0.1 bits. Some promoters lack a −35 region, but have a 6.7 ± 0.2 bit extended −10, almost the same information as the bipartite promoter. These results and similarities between the contacts in the extended −10 binding and the −35 suggest that the flexible bipartite σ factor evolved from a simpler polymerase. Binding predicted by the bipartite model is enriched around 35 bases upstream of the translational start. This distance is the smallest 5′ mRNA leader necessary for ribosome binding, suggesting that selective pressure minimizes transcript length. The promoter model was combined with models of the transcription factors Fur and Lrp to locate new promoters, to quantify promoter strengths, and to predict activation and repression. Finally, the DNA-bending proteins Fis, H-NS and IHF frequently have sites within one DNA persistence length from the −35, so bending allows distal activators to reach the polymerase.

Accelerated formation of α-synuclein oligomers by concerted action of the 20S proteasome and familial Parkinson mutations

A hallmark of Parkinson disease (PD) is the formation of intracellular protein inclusions called Lewy bodies that also contain mitochondria. α-Synuclein (αSyn) is a major protein component of Lewy bodies, where it is in an amyloid conformation and a significant fraction is truncated by poorly understood proteolytic events. Previously, we demonstrated that the 20S proteasome cleaves αSyn in vitro to produce fragments like those observed in Lewy bodies and that the fragments accelerate the formation of amyloid fibrils from full-length αSyn. Three point mutations in αSyn are associated with early-onset familial PD: A30P, E46K, and A53T. However, these mutations have very different effects on the amyloidogenicity and vesicle-binding activity of αSyn, suggesting neither of these processes directly correlate with neurodegeneration. Here, we evaluate the effect of the disease-associated mutations on the fragmentation, conformation, and association reactions of αSyn in the presence of the 20S proteasome and liposomes. The 20S proteasome produced the C-terminal fragments from both the mutant and wildtype αSyn. These truncations accelerated fibrillization of all α-synucleins, but again there was no clear correlation between the PD-associated mutations and amyloid formation in the presence of liposomes. Recent data suggests that cellular toxicity is caused by a soluble oligomeric species, which is a precursor to the amyloid form and is immunologically distinguishable from both soluble monomeric and amyloid forms of αSyn. Notably, the rate of formation of the soluble, presumptively cytotoxic oligomers correlated with the disease-associated mutations when both 20S proteasome and liposomes were present. Under these conditions, the wildtype protein was also cleaved and formed the oligomeric structures, albeit at a slower rate, suggesting that 20S-mediated truncation of αSyn may play a role in sporadic PD as well. Evaluation of the biochemical reactions of the PD-associated α-synuclein mutants in our in vitro system provides insight into the possible pathogenetic mechanism of both familial and sporadic PD.

Abnormal Neurites Containing C-Terminally Truncated α-Synuclein Are Present in Alzheimer's Disease without Conventional Lewy Body Pathology

The pathological hallmark of Parkinsons disease and diffuse Lewy body disease (DLBD) is the aggregation of α-synuclein (α-syn) in the form of Lewy bodies and Lewy neurites. Patients with both Alzheimers disease (AD) and cortical Lewy pathology represent the Lewy body variant of AD (LBV) and constitute 25% of AD cases. C-terminally truncated forms of α-syn enhance the aggregation of α-syn in vitro. To investigate the presence of C-terminally truncated α-syn in DLBD, AD, and LBV, we generated and validated polyclonal antibodies to truncated α-syn ending at residues 110 (α-syn110) and 119 (α-syn119), two products of 20S proteosome-mediated endoproteolytic cleavage. Double immunofluorescence staining of the cingulate cortex showed that α-syn110 and α-syn140 (full-length) aggregates were not colocalized in LBV. All aggregates containing α-syn140 also contained α-syn119; however, some aggregates contained α-syn119 without α-syn140, suggesting that α-syn119 may stimulate aggregate formation. Immunohistochemistry and image analysis of tissue microarrays of the cingulate cortex from patients with DLBD (n = 27), LBV (n = 27), and AD (n = 19) and age-matched controls (n = 15) revealed that AD is also characterized by frequent abnormal neurites containing α-syn119. Notably, these neurites did not contain α-syn ending at residues 110 or 122-140. The presence of abnormal neurites containing α-syn119 in AD without conventional Lewy pathology suggests that AD and Lewy body disease may be more closely related than previously thought.

The tenacious recognition of yeast telomere sequence by Cdc13 is fully exerted by a single OB-fold domain

Cdc13, the telomere end-binding protein from Saccharomyces cerevisiae, is a multidomain protein that specifically binds telomeric single-stranded DNA (ssDNA) with exquisitely high affinity to coordinate telomere maintenance. Recent structural and genetic data have led to the proposal that Cdc13 is the paralog of RPA70 within a telomere-specific RPA complex. Our understanding of Cdc13 structure and biochemistry has been largely restricted to studies of individual domains, precluding analysis of how each domain influences the activity of the others. To better facilitate a comparison to RPA70, we evaluated the ssDNA binding of full-length S. cerevisiae Cdc13 to its minimal substrate, Tel11. We found that, unlike RPA70 and the other known telomere end-binding proteins, the core Cdc13 ssDNA-binding activity is wholly contained within a single tight-binding oligosaccharide/oligonucleotide/oligopeptide binding (OB)-fold. Because two OB-folds are implicated in dimerization, we also evaluated the relationship between dimerization and ssDNA-binding activity and found that the two activities are independent. We also find that Cdc13 binding exhibits positive cooperativity that is independent of dimerization. This study reveals that, while Cdc13 and RPA70 share similar domain topologies, the corresponding domains have evolved different and specialized functions.

Practical strategies for the evaluation of high-affinity protein/nucleic acid interactions.

The quantitative evaluation of binding interactions between proteins and nucleic acids is highly sensitive to a variety of experimental conditions. Optimization of these conditions is critical for obtaining high quality, reproducible data, particularly in the context of very high affinity interactions. Here, we discuss the practical considerations involved in optimizing the apparent binding constant of an interaction as measured by two common quantitative assays, electrophoretic mobility shift assay and double-filter binding when measuring extremely tight protein/nucleic acid interactions with sub-nanomolar binding affinities. We include specific examples from two telomere end-binding protein systems, Schizo -saccharomyces pombe Pot1 and Saccharomyces cerevisiae Cdc13, to demonstrate potential experimental pitfalls and some useful strategies for optimization.

Optical and X‐ray Properties of the Swift BAT‐detected AGN

The Swift Gamma‐Ray Burst satellite has detected a largely unbiased towards absorption sample of local ( ≈0.03) AGN, based solely on their 14–195 keV flux. In the first 9 months of the survey, 153 AGN sources were detected. The X‐ray properties in the 0.3–10 keV band have been compiled and presented based on analyses with XMM‐Newton, Chandra, Suzaku, and the Swift XRT (Winter et al. 2009). Additionally, we have compiled a sub‐sample of sources with medium resolution optical ground‐based spectra from the SDSS or our own observations at KPNO. In this sample of 60 sources, we have classified the sources using standard emission line diagnostic plots, obtained masses for the broad line sources through measurement of the broad Hβ emission line, and measured the [OIII] 5007A luminosity of this sample. Based on continuum fits to the intrinsic absorption features, we have obtained clues about the stellar populations of the host galaxies. We now present the highlights of our X‐ray and optical studies of this uniq...