Adele Rowley

Proteomics Characterization of Abundant Golgi Membrane Proteins

A mass spectrometric analysis of proteins partitioning into Triton X-114 from purified hepatic Golgi apparatus (84% purity by morphometry, 122-fold enrichment over the homogenate for the Golgi marker galactosyl transferase) led to the unambiguous identification of 81 proteins including a novel Golgi-associated protein of 34 kDa (GPP34). The membrane protein complement was resolved by SDS-polyacrylamide gel electrophoresis and subjected to a hierarchical approach using delayed extraction matrix-assisted laser desorption ionization mass spectrometry characterization by peptide mass fingerprinting, tandem mass spectrometry to generate sequence tags, and Edman sequencing of proteins. Major membrane proteins corresponded to known Golgi residents, a Golgi lectin, anterograde cargo, and an abundance of trafficking proteins including KDEL receptors, p24 family members, SNAREs, Rabs, a single ARF-guanine nucleotide exchange factor, and two SCAMPs. Analytical fractionation and gold immunolabeling of proteins in the purified Golgi fraction were used to assess the intra-Golgi and total cellular distribution of GPP34, two SNAREs, SCAMPs, and the trafficking proteins GBF1, BAP31, and α2P24 identified by the proteomics approach as well as the endoplasmic reticulum contaminant calnexin. Although GPP34 has never previously been identified as a protein, the localization of GPP34 to the Golgi complex, the conservation of GPP34 from yeast to humans, and the cytosolically exposed location of GPP34 predict a role for a novel coat protein in Golgi trafficking.

Chronic treatment with a novel γ-secretase modulator, JNJ-40418677, inhibits amyloid plaque formation in a mouse model of Alzheimer's disease

γ‐Secretase modulators represent a promising therapeutic approach for Alzheimers disease (AD) because they selectively decrease amyloid β 42 (Aβ42), a particularly neurotoxic Aβ species that accumulates in plaques in the brains of patients with AD. In the present study, we describe the in vitro and in vivo pharmacological properties of a potent novel γ‐secretase modulator, 2‐(S)‐(3,5‐bis(4‐(trifluoromethyl)phenyl)phenyl)‐4‐methylpentanoic acid (JNJ‐40418677).

Prion-mediated diversity in yeast

In their natural environment, yeast such as Saccharomyces cerevisiae encounter wildly fluctuating external conditions and must be able to adapt rapidly to their changing environment. True and Lindquist1xA yeast prion provides a mechanism for genetic variation and phenotypic diversity. True, H.L. and Lindquist, S.L. Nature. 2000; 407: 477–483Crossref | PubMed | Scopus (406)See all References1 now propose that a conserved prion-mediated mechanism might allow a yeast cell to modulate its phenotype reversibly by taking advantage of pre-existing, but normally silent, genetic variation.Yeast prions are heritable protein elements that affect the fidelity of translation-termination. The prion protein, encoded by the yeast SUP35 gene, is a translation termination factor that can exist in either a normal [psi−] or subverted prion [PSI+] state. Once corrupted, [PSI+] Sup35 can recruit new Sup35 protein to an insoluble prion complex, thereby perpetuating the [PSI+] state. In its [PSI+] state, Sup35p is essentially sequestered from the rest of the translation-termination apparatus, resulting in read-through of stop codons in reporter genes and also (presumably) in some endogenous transcripts. Thus, cells with the same genotype can potentially exhibit different phenotypes depending on the state of the Sup35 protein.Previously, only anecdotal evidence suggested that [PSI] status influences cell growth and survival. Now however, in a series of elegantly simple ‘fitness’ tests, True and Lindquist have compared the growth of freshly generated isogenic [PSI+] and [psi−] strains, uncovering a widespread influence on cell growth under different environmental conditions in different genetic backgrounds. The observations do not fit a tidy pattern; in some cases [PSI+] cells grew better, in other cases the [psi−] state prevailed, and this was often completely different in divergent genetic backgrounds. [PSI] status also resulted in pronounced differences in colony morphology.These experiments demonstrate clearly that [PSI+] increases phenotypic diversity without permanent changes to the genome, changes that might not always be advantageous. Thus, True and Lindquist propose that [PSI+]-induced diversity offers the opportunity of rapid adaptation to a new environment. As cells switch frequently between [PSI+] and [psi−] states, such changes would be readily reversible, allowing a population of cells to rapidly re-adapt to their original environmental niche if necessary. The molecular basis of [PSI+]-facilitated diversity is unclear but presumably involves translational read-through at internal stop codons within nonessential, perhaps duplicated, target gene(s) or alternatively at stop codons at the 39 end of genes. The consequences of such read-through events could be variable and gene dependent, perhaps affecting protein or mRNA stability, or in some cases resulting in the production of extended proteins. Many questions remain and it is clear that the study of what was originally known as an obscure genetic phenomenon in yeast still has much to teach us.

Identification of the tau interactome in mouse brain

E2 brought these changes to near normalcy.Conclusions: It can therefore be concluded that E2’s beneficial effects seemed to arise from its antilipofuscin, antioxidant and antilipidperoxidative effects, implying an overall anti-aging action. The results of this study will be useful for pharmacological modification of the aging process and applying new strategies for control of age related disorders.

Identification of proteins that form complexes with tau in mouse brain

Pin1 knockout in mice causes both tau and Ab-related pathologies as well as neurodegeneration in an age-dependent manner. By contrast, transgenic Pin1 overexpression in postnatal neurons prevents the development of AD in mice. Together with the findings that Pin1 is inhibited by multiple mechanisms in human AD, these results indicate that Pin1 is pivotal for protecting against AD. However, it is not clear how Pin1 protects against tau and Abeta-related pathologies and whether Pin1 affects onset of AD. Methods: We have used multiple approaches, including generating conformation-specific antibodies, using NMR, cell cultures, mouse models, human samples and genetic association studies. Results: We found that Pin1 promoted cis to trans isomerization of the pThr231-Pro motif in tau and pThr668-Pro motif in APP. Without functional Pin1, both tau and APP were accumulated in the cis conformation, which led to increased tau phosphorylation and reduced tau degradation, as well as increased APP internalization, resulting in amyloidogenic APP processing and Abeta production. The significance of these findings is further substantiated by our identification of a functional polymorphism in the Pin1 promoter that was significantly associated with a 3-year delay in the average age-at-onset (AAO) of late-onset AD in a Chinese population. This Pin1 polymorphism completely abolished the ability of a brain-selective transcription factor to bind and suppress the Pin1 promoter and to reduce Pin1 protein levels. Conclusions: We have shown that Pin1 is a pivotal new enzyme that protects against tau and Abeta-related pathologies in AD by controlling the conformation of tau and APP after phosphorylation and also identify a novel Pin1 regulation that is critical in determining onset of AD. These results further demonstrate that Pin1 is an attractive novel therapeutic target to treating AD or delay its onset.

Effects of γ- secretase Modulation in Cellular and Animal Models of Abeta Secretion

83.36 10 and 60.66 9.4 (p< 0.0001); Linguistic Comprehension: 98.76 5.3 and 91.16 6.6 (p1⁄4 0.0001), and Visuospatial Construction: 24.66 1.7 and 19.26 4.2 (p < 0.0001). Total score of ABCD was 83.26 4.8 for controls and 67.2 6 6.7 in the VaD group (p < 0.0001). Linguistic Expression tasks were more effective in discriminating VaD patients from controls than Linguistic Comprehension tasks. Conclusions: The ABCD is a useful tool to identify and discriminate linguistic performance of VaD patients from normal elderly. Executive dysfunction (including working memory), and attentional impairment, may have influenced the patients’ linguistic performance.

Genome-wide screen for protein-protein interaction in yeast

Using the yeast 2 hybrid system, Uetz et al. 1 report the first genome-wide study in Saccharomyces cerevisiae to investigate protein‐protein interactions. The yeast two-hybrid system exploits the observation that the DNAbinding and transcriptional-activation domains of a transcription factor such as Gal4 are separable, and that they can activate transcription, even when expressed on different fusion proteins, provided that they are brought into close proximity at the promotor of a suitable reporter gene. This is achieved by fusing each domain to one or other of a pair of proteins that physically interact and then combining these constructs in the same yeast cell. Furthermore, because yeast cells exist in two complementary mating types, it is relatively easy to introduce DNAbinding and activation-domain fusions to one another via a simple transformant-mixing strategy. The authors have used two approaches to assay the ability of DNA-binding domain fusions to interact with all possible activation-domain fusions. In the first, 192 individual DNA-binding-domain fusions were tested against almost all potential activation-domain constructs in an arrayed library format; in the second, DNA-binding-domain fusions were mixed with pooled activation-domain constructs. The first approach, while of lower throughput, resulted in a higher number of interactions/bait. Overall, 957 putative interactions (109 of which had been previously discovered), involving 1004 proteins, were identified. To manipulate the large data set, Uetz et al. developed bioinformatics tools to view protein connections and to integrate data with other pertinent information, for example homologues in other organisms. The results of this study clearly tether many previously unassigned genes to networks of interacting proteins and reveal new connections within and between pathways, even alluding to the existence of previously unidentified pathways. Combined with other genome-wide efforts and an existing wealth of genetic information, this study marks another milestone in progress towards a complete understanding of regulatory mechanisms in this organism.

Ku—that's an interesting protein

Abstract Yeast Ku as a regulator of chromosomal DNA end structure Gravel, S., Larrivee, M., Labrecque, P. and Wellinger, R.J. Science 280, 741–744 Components of the Ku-dependent non-homologous end-joining pathway are involved in telomeric length maintenance and telomeric silencing Boulton, S.J. and Jackson, S.P. EMBO J . 17, 1819–1828