Rana palustris

Number of Pages: 3Integrative BiologyGeological Science

Analysis of polyubiquitin conjugates reveals that the Rpn10 substrate receptor contributes to the turnover of multiple proteasome targets

The polyubiquitin receptor Rpn10 targets ubiquitylated Sic1 to the 26S proteasome for degradation. In contrast, turnover of at least one ubiquitin-proteasome system (UPS) substrate, CPY*, is impervious to deletion of RPN10. To distinguish whether RPN10 is involved in the turnover of only a small set of cell cycle regulators that includes Sic1 or plays a more general role in the UPS, we sought to develop a general method that would allow us to survey the spectrum of ubiquitylated proteins that selectively accumulate in rpn10 cells. Polyubiquitin conjugates from yeast cells that express hexahistidine-tagged ubiquitin (H6-ubiquitin) were first enriched on a polyubiquitin binding protein affinity resin. This material was then denatured and subjected to IMAC to retrieve H6-ubiquitin and proteins to which it may be covalently linked. Using this approach, we identified 127 proteins that are candidate substrates for the 26S proteasome. We then sequenced ubiquitin conjugates from cells lacking Rpn10 (rpn10) and identified 54 proteins that were uniquely recovered from rpn10 cells. These include two known targets of the UPS, the cell cycle regulator Sic1 and the transcriptional activator Gcn4. Our approach of comparing the ubiquitin conjugate proteome in wild-type and mutant cells has the resolving power to identify even an extremely inabundant transcriptional regulatory protein and should be generally applicable to mapping enzyme substrate networks in the UPS

Reactions of Large Groups of Caribou to a Pipeline Corridor on the Arctic Coastal Plain of Alaska

Two large groups of mosquito-harassed caribou (Rangifer tarandus granti) were followed for 8-12 h as they repeatedly attempted to cross an elevated pipeline in the Kuparuk Development Area near Prudhoe Bay, Alaska. In 1981, 46% of a group of 917 eventually crossed beneath elevated portions of the pipeline in 26 separate attempts, 13% crossed a section of buried pipe in two attempts, 22% trotted parallel to the pipeline for 32 km and did not cross, and 19% separated from the group and were not accounted for. In 1982, 26% of a group of 655 crossed under elevated portions of the pipeline in 36 attempts, 37% crossed at a buried section in one attempt, and 37% left the main group and could not be accounted for. The majority of crossing attempts occured near intersections of lakes with the road/pipeline complex, but crossing success was highest at a section of buried pipe isolated from the road traffic.Key words: caribou, pipeline, petroleum development, insect harassment, Kuparuk Oil FieldMots clés: caribou, pipe-line, développement pétrolifère, harcèlement par les moustiques, champ pétrolifère Kuparu

A common mechanism of proteasome impairment by neurodegenerative disease-associated oligomers

Protein accumulation and aggregation with a concomitant loss of proteostasis often con- tribute to neurodegenerative diseases, and the ubiquitin–proteasome system plays a major role in protein degradation and proteostasis. Here, we show that three different proteins from Alzheimer’s, Parkinson’s, and Huntington’s disease that misfold and oligomerize into a shared three-dimensional structure potently impair the proteasome. This study indicates that the shared conformation allows these oligomers to bind and inhibit the proteasome with low nanomolar affinity, impairing ubiquitin-dependent and ubiquitin-independent proteasome function in brain lysates. Detailed mechanistic analysis demonstrates that these oligomers inhibit the 20S proteasome through allosteric impairment of the substrate gate in the 20S core particle, preventing the 19S regulatory particle from injecting substrates into the degradation chamber. These results provide a novel molecular model for oligomer-driven impairment of proteasome function that is relevant to a variety of neurodegenerative dis- eases, irrespective of the specific misfolded protein that is involved

The steady-state repertoire of human SCF Ubiquitin ligase complexes does not require ongoing Nedd8 conjugation

The human genome encodes 69 different F-box proteins (FBPs), each of which can potentially assemble with Skp1-Cul1-RING to serve as the substrate specificity subunit of an SCF ubiquitin ligase complex. SCF activity is switched on by conjugation of the ubiquitin- like protein Nedd8 to Cul1. Cycles of Nedd8 conjugation and deconjugation acting in conjunction with the Cul1-sequestering factor Cand1 are thought to control dynamic cycles of SCF assembly and disassembly, which would enable a dynamic equilibrium between the Cul1- RING catalytic core of SCF and the cellular repertoire of FBPs. To test this hypothesis, we determined the cellular composition of SCF complexes and evaluated the impact of Nedd8 conjugation on this steady-state. At least 42 FBPs assembled with Cul1 in HEK 293 cells, and the levels of Cul1-bound FBPs varied by over two orders of magnitude. Unexpectedly, quantitative mass spectrometry revealed that blockade of Nedd8 conjugation led to a modest increase, rather than a decrease, in the overall level of most SCF complexes. We suggest that multiple mechanisms including FBP dissociation and turnover cooperate to maintain the cellular pool of SCF ubiquitin ligases

A putative stimulatory role for activator turnover in gene expression

The ubiquitin–proteasome system (UPS) promotes the destruction of target proteins by attaching to them a ubiquitin chain that is recognized by the 26S proteasome. The UPS influences most cellular processes, and its targets include transcriptional activators that are primary determinants of gene expression. Emerging evidence indicates that non-proteolytic functions of the UPS might stimulate transcriptional activity. Here we show that the proteolysis of some transcriptional activators by the UPS can stimulate their function. We focused on the role of UPS-dependent proteolysis in the function of inducible transcriptional activators in yeast, and found that inhibition of the proteasome reduced transcription of the targets of the activators Gcn4, Gal4 and Ino2/4. In addition, mutations in SCF^(Cdc4), the ubiquitin ligase for Gcn4 (ref. 5), or mutations in ubiquitin that prevent degradation, also impaired the transcription of Gcn4 targets. These transcriptional defects were manifested despite the enhanced abundance of Gcn4 on cognate promoters. Proteasome inhibition also decreased the association of RNA polymerase II with Gcn4, Gal4 and Ino2/4 targets, as did mutations in SCFCdc4 for Gcn4 targets. Expression of a stable phospho-site mutant of Gcn4 (ref. 7) or disruption of the kinases that target Gcn4 for turnover alleviated the sensitivity of Gcn4 activity to defects in the UPS

Central Arctic Caribou and Petroleum Development: Distributional, Nutritional, and Reproductive Implications

We synthesize findings from cooperative research on effects of petroleum development on caribou (Rangifer tarandus granti) of the Central Arctic Herd (CAH). The CAH increased from about 6000 animals in 1978 to 23000 in 1992, declined to 18 000 by 1995, and again increased to 27 000 by 2000. Net calf production was consistent with changes in herd size. In the Kuparuk Development Area (KDA), west of Prudhoe Bay, abundance of calving caribou was less than expected within 4 km of roads and declined exponentially with road density. With increasing infrastructure, high-density calving shifted from the KDA to inland areas with lower forage biomass. During July and early August, caribou were relatively unsuccessful in crossing road/pipeline corridors in the KDA, particularly when in large, insect-harassed aggregations; and both abundance and movements of females were lower in the oil field complex at Prudhoe Bay than in other areas along the Arctic coast. Female caribou exposed to petroleum development west of the Sagavanirktok River may have consumed less forage during the calving period and experienced lower energy balance during the midsummer insect season than those under disturbance-free conditions east of the river. The probable consequences were poorer body condition at breeding and lower parturition rates for western females than for eastern females (e.g., 1988–94: 64% vs. 83% parturient, respectively; p = 0.003), which depressed the productivity of the herd. Assessments of cumulative effects of petroleum development on caribou must incorporate the complex interactions with a variable natural environment.On a procédé à une synthèse des résultats de travaux de recherche coopérative concernant les effets de l’exploitation pétrolière sur le caribou (Rangifer tarandus granti) formant la harde du centre de l’Arctique (HCA). La population de celle-ci est passée de 6000 têtes en 1978 à 23 000 en 1992, puis a diminué à 18 000 en 1995 pour augmenter de nouveau à 27 000 en 2000. La production nette des veaux allait de pair avec les changements dans la taille de la harde. Dans la zone de développement de Kuparuk (KDA), située à l’ouest de Prudhoe Bay, l’abondance des caribous qui mettaient bas était inférieure à celle prévue dans une bande de 4 km de part et d’autre des routes, et elle déclinait de façon exponentielle avec la densité routière. Avec une augmentation des infrastructures, on assistait à un déplacement du vêlage à forte densité de la KDA vers des zones de l’intérieur ayant une biomasse de fourrage moins importante. Durant juillet et au début d’août, il était assez rare que les caribous réussissent à traverser les corridors routiers/pipeliniers dans la KDA, surtout lorsqu’ils formaient de vastes agrégations harcelées par les insectes; l’abondance de même que les déplacements des femelles étaient en outre moindres au sein du complexe pétrolier de Prudhoe Bay qu’à d’autres endroits situés le long du rivage arctique. Il est possible que les femelles qui étaient exposées à l’exploitation pétrolière à l’ouest de la rivière Sagavanirktok aient consommé moins de fourrage au cours de la période de vêlage et que, durant la saison des insectes au milieu de l’été, elles aient connu une balance énergétique inférieure à celle des femelles vivant sans perturbations à l’est de la rivière. Les conséquences probables étaient un état corporel de qualité inférieure au moment de l’accouplement, et des taux de parturition plus faibles pour les femelles situées à l’ouest que pour celles situées à l’est (p. ex., de 1988 à 1994: 64 % c. 83 % de parturientes respectivement: p = 0,003), faisant ainsi baisser la productivité de la harde. Les évaluations des effets cumulatifs de l’exploitation pétrolière sur le caribou doivent intégrer les interactions complexes avec un environnement naturel variable

Cdc48/p97 Mediates UV-Dependent Turnover of RNA Pol II

Cdc48/p97 is an essential ATPase whose role in targeting substrates to the ubiquitin-proteasome system (UPS) remains unclear. Existing models posit that Cdc48 acts upstream of UPS receptors. To address this hypothesis, we examined the association of ubiquitin (Ub) conjugates with 26S proteasomes. Unexpectedly, proteasomes isolated from cdc48 mutants contain high levels of Ub conjugates, and mass spectrometry identified numerous nonproteasomal proteins, including Rpb1, the largest subunit of RNA Pol II. UV-induced turnover of Rpb1 depends upon Cdc48-Ufd1-Npl4, Ubx4, and the uncharacterized adaptor Ubx5. Ubiquitinated Rpb1, proteasomes, and Cdc48 accumulate on chromatin in UV-treated wild-type cells, and the former two accumulate to higher levels in mutant cells, suggesting that degradation of Rpb1 is facilitated by Cdc48 at sites of stalled transcription. These data reveal an intimate coupling of function between proteasomes and Cdc48 that we suggest is necessary to sustain processive degradation of unstable subunits of some macromolecular protein complexes

A Hubble Space Telescope Imaging Survey of Nearby Active Glactic Nuclei

We obtained 500-second F606W WFPC2 images of 256 of the nearest (z<0.035) Seyfert 1,Seyfert 2, and starburst galaxies. Less than 10% show tidal features or multiple nuclei. The incidence of inner starburst rings is about 10% in both classes of Sy galaxies. In contrast, galaxies with H II region emission line spectra appear substantially more irregular because of their much higher specific rates of star formation. An unresolved central continuum source in our HST images is a virtually perfect indicator of a Sy1 spectrum. 52% of these Sy1 point sources are saturated in our images; we use their wings to estimate their magnitudes. The converse is not however true, as over a third of Sy's with direct spectroscopic evidence for broad Balmer wings show no nuclear point source. Like the Sy2's, they have central surface brightnesses consistent with those expected for the bulges of normal galaxies. The frequency of bars in Sy1's and 2's and non-Sys are the same. The Sy2 galaxies are significantly more likely to show nuclear dust absorption, especially in lanes and patches which are irregular or reach close to the nucleus. The difference cannot be explained by different average redshifts or selection techniques. This is confirmed by our morphology classifications, which show that Sy1 nuclei reside in earlier type galaxies than Sy2 nuclei. This intrinsic difference in host galaxy properties may undermine the strong unification hypothesis for Sy galaxies that they appear different due to the orientation of their central engine. The excess galactic dust we see in Sy2's may cause substantial absorption which obscures their hypothesized broad emission-line regions and central nonstellar continua. This galactic dust could produce much of the absorption in Sy2 nuclei which had instead been attributed to a thick dusty accretion torus.Comment: The text of the paper is 23 pages (ms.tex), there are 8 tables, and 9 figures. Figures 1, 2, and 3 are the image gallery (45 pages) and are NOT included here. They can be ftp'ed from ftp.astro.ucla.edu. Log in as anonymous and give your e-mail address as the password. The images are in the /pub/submit/vg/AGNgallery . Figures 4-9 are in eps format and are included here and can be printed using the lpr command in unix system

Redistribution of Calving Caribou in Response to Oil Field Development on the Arctic Slope of Alaska

Aerial surveys were conducted annually in June 1978-87 near Prudhoe Bay, Alaska, to determine changes in the distribution of calving caribou (Rangifer tarandus granti) that accompanied petroleum-related development. With construction of an oil field access road through a calving concentration area, mean caribou density (no./sq. km) decreased from 1.41 to 0.31 (P=0.05) with 1 km and increased from 1.41 to 4.53 (P=0.04) 5-6 km from the road. Concurrently, relative caribou use of the adjacent area declined (P&lt;0.02), apparently in response to increasing surface development. We suggest that perturbed distribution associated with roads reduced the capacity of the nearby area to sustain parturient females and that insufficient spacing of roads may have depressed overall calving activity. Use of traditional calving grounds and of certain areas therein appears to favor calf survival, principally through lower predation risk and improved foraging conditions. Given the possible loss of those habitats through displacement and the crucial importance of the reproductive process, a cautious approach to petroleum development on the Arctic Slope is warranted.Key words: Alaska, calving, caribou, disturbance, oil fieldR&Eacute;SUM&Eacute;. Des photographies a&eacute;riennes ont &eacute;t&eacute; prises au mois de juin chaque ann&eacute;e de 1978 &agrave; 1987 pr&egrave;s de la baie Prudhoe en Alaska, en vue de d&eacute;terminer les changements dans la distribution des caribous (Rangifer tarandus granti) qui mettaient bas, changements accompagnant les projets de mise en valeur reli&eacute;s au p&eacute;trole. Avec la construction d&rsquo;une route d&rsquo;acc&egrave;s &agrave; un champ p&eacute;trolif&egrave;re &agrave; travers une zone importante de mise bas, la densit&eacute; moyenne du caribou (nombre d&rsquo;individus/km2) diminuait de 1,41 &agrave; 0,31 (P = 0,OS) &agrave; moins d&rsquo;1 km de la route et augmentait de 1,41 &agrave; 4,53 (P = 0,04) &agrave; une distance de 516 km de la route. En m&ecirc;me temps, l&rsquo;utilisation relative de la zone adjacente par le caribou diminuait (P &lt; 0,02), apparemment enr&eacute;ponse &agrave; l&rsquo;augmentation de la mise en valeur de surface. On sugg&egrave;re que la perturbation de la distribution associ&eacute;e &agrave; la construction de routes diminuait la capacit&eacute; de la zone environnante &agrave; supporter des femelles parturientes et qu&rsquo;un espacement insuffisant des routes aurait pu faire chuter l&rsquo;activit&eacute; g&eacute;n&eacute;rale de mise bas. L&rsquo;utilisation de terrains traditionnels de mise bas ainsi que de certaines zones &agrave; l&rsquo;int&eacute;rieur de ces terrains semble favoriser la survie des veaux, en particulier gr&acirc;ce &agrave; une baisse de la pr&eacute;dation et &agrave; de meilleures conditions de p&acirc;turage. &Eacute;tant donn&eacute; que le caribou pourrait &ecirc;tre &eacute;vinc&eacute; de cet habitat et vu l&rsquo;importance cruciale du processus de reproduction, on recommande une approche prudente &agrave; l&rsquo;exploitation du p&eacute;trole sur le versant arctique.Mots cl&eacute;s: Alaska, mise bas, caribou, perturbation, champ p&eacute;trolif&egrave;r