Prion protein repeat expansion results in increased aggregation and reveals phenotypic variability

Mammalian prion diseases are fatal neurodegenerative disorders dependent on the prion protein PrP. Expansion of the oligopeptide repeats (ORE) found in PrP is associated with inherited prion diseases. Patients with ORE frequently harbor PrP aggregates, but other factors may contribute to pathology, as they often present with unexplained phenotypic variability. We created chimeric yeast-mammalian prion proteins to examine the influence of the PrP ORE on prion properties in yeast. Remarkably, all chimeric proteins maintained prion characteristics. The largest repeat expansion chimera displayed a higher propensity to maintain a self-propagating aggregated state. Strikingly, the repeat expansion conferred increased conformational flexibility, as observed by enhanced phenotypic variation. Furthermore, the repeat expansion chimera displayed an increased rate of prion conversion, but only in the presence of another aggregate, the [RNQ(+)] prion. We suggest that the PrP ORE increases the conformational flexibility of the prion protein, thereby enhancing the formation of multiple distinct aggregate structures and allowing more frequent prion conversion. Both of these characteristics may contribute to the phenotypic variability associated with PrP repeat expansion diseases

The state of the Martian climate

60°N was +2.0°C, relative to the 1981–2010 average value (Fig. 5.1). This marks a new high for the record. The average annual surface air temperature (SAT) anomaly for 2016 for land stations north of starting in 1900, and is a significant increase over the previous highest value of +1.2°C, which was observed in 2007, 2011, and 2015. Average global annual temperatures also showed record values in 2015 and 2016. Currently, the Arctic is warming at more than twice the rate of lower latitudes

Disease-Associated Mutant Ubiquitin Causes Proteasomal Impairment and Enhances the Toxicity of Protein Aggregates

Protein homeostasis is critical for cellular survival and its dysregulation has been implicated in Alzheimer's disease (AD) and other neurodegenerative disorders. Despite the growing appreciation of the pathogenic mechanisms involved in familial forms of AD, much less is known about the sporadic cases. Aggregates found in both familial and sporadic AD often include proteins other than those typically associated with the disease. One such protein is a mutant form of ubiquitin, UBB+1, a frameshift product generated by molecular misreading of a wild-type ubiquitin gene. UBB+1 has been associated with multiple disorders. UBB+1 cannot function as a ubiquitin molecule, and it is itself a substrate for degradation by the ubiquitin/proteasome system (UPS). Accumulation of UBB+1 impairs the proteasome system and enhances toxic protein aggregation, ultimately resulting in cell death. Here, we describe a novel model system to investigate how UBB+1 impairs UPS function and whether it plays a causal role in protein aggregation. We expressed a protein analogous to UBB+1 in yeast (Ubext) and demonstrated that it caused UPS impairment. Blocking ubiquitination of Ubext or weakening its interactions with other ubiquitin-processing proteins reduced the UPS impairment. Expression of Ubext altered the conjugation of wild-type ubiquitin to a UPS substrate. The expression of Ubext markedly enhanced cellular susceptibility to toxic protein aggregates but, surprisingly, did not induce or alter nontoxic protein aggregates in yeast. Taken together, these results suggest that Ubext interacts with more than one protein to elicit impairment of the UPS and affect protein aggregate toxicity. Furthermore, we suggest a model whereby chronic UPS impairment could inflict deleterious consequences on proper protein aggregate sequestration

State of the climate in 2018

In 2018, the dominant greenhouse gases released into Earth’s atmosphere—carbon dioxide, methane, and nitrous oxide—continued their increase. The annual global average carbon dioxide concentration at Earth’s surface was 407.4 ± 0.1 ppm, the highest in the modern instrumental record and in ice core records dating back 800 000 years. Combined, greenhouse gases and several halogenated gases contribute just over 3 W m−2 to radiative forcing and represent a nearly 43% increase since 1990. Carbon dioxide is responsible for about 65% of this radiative forcing. With a weak La Niña in early 2018 transitioning to a weak El Niño by the year’s end, the global surface (land and ocean) temperature was the fourth highest on record, with only 2015 through 2017 being warmer. Several European countries reported record high annual temperatures. There were also more high, and fewer low, temperature extremes than in nearly all of the 68-year extremes record. Madagascar recorded a record daily temperature of 40.5°C in Morondava in March, while South Korea set its record high of 41.0°C in August in Hongcheon. Nawabshah, Pakistan, recorded its highest temperature of 50.2°C, which may be a new daily world record for April. Globally, the annual lower troposphere temperature was third to seventh highest, depending on the dataset analyzed. The lower stratospheric temperature was approximately fifth lowest. The 2018 Arctic land surface temperature was 1.2°C above the 1981–2010 average, tying for third highest in the 118-year record, following 2016 and 2017. June’s Arctic snow cover extent was almost half of what it was 35 years ago. Across Greenland, however, regional summer temperatures were generally below or near average. Additionally, a satellite survey of 47 glaciers in Greenland indicated a net increase in area for the first time since records began in 1999. Increasing permafrost temperatures were reported at most observation sites in the Arctic, with the overall increase of 0.1°–0.2°C between 2017 and 2018 being comparable to the highest rate of warming ever observed in the region. On 17 March, Arctic sea ice extent marked the second smallest annual maximum in the 38-year record, larger than only 2017. The minimum extent in 2018 was reached on 19 September and again on 23 September, tying 2008 and 2010 for the sixth lowest extent on record. The 23 September date tied 1997 as the latest sea ice minimum date on record. First-year ice now dominates the ice cover, comprising 77% of the March 2018 ice pack compared to 55% during the 1980s. Because thinner, younger ice is more vulnerable to melting out in summer, this shift in sea ice age has contributed to the decreasing trend in minimum ice extent. Regionally, Bering Sea ice extent was at record lows for almost the entire 2017/18 ice season. For the Antarctic continent as a whole, 2018 was warmer than average. On the highest points of the Antarctic Plateau, the automatic weather station Relay (74°S) broke or tied six monthly temperature records throughout the year, with August breaking its record by nearly 8°C. However, cool conditions in the western Bellingshausen Sea and Amundsen Sea sector contributed to a low melt season overall for 2017/18. High SSTs contributed to low summer sea ice extent in the Ross and Weddell Seas in 2018, underpinning the second lowest Antarctic summer minimum sea ice extent on record. Despite conducive conditions for its formation, the ozone hole at its maximum extent in September was near the 2000–18 mean, likely due to an ongoing slow decline in stratospheric chlorine monoxide concentration. Across the oceans, globally averaged SST decreased slightly since the record El Niño year of 2016 but was still far above the climatological mean. On average, SST is increasing at a rate of 0.10° ± 0.01°C decade−1 since 1950. The warming appeared largest in the tropical Indian Ocean and smallest in the North Pacific. The deeper ocean continues to warm year after year. For the seventh consecutive year, global annual mean sea level became the highest in the 26-year record, rising to 81 mm above the 1993 average. As anticipated in a warming climate, the hydrological cycle over the ocean is accelerating: dry regions are becoming drier and wet regions rainier. Closer to the equator, 95 named tropical storms were observed during 2018, well above the 1981–2010 average of 82. Eleven tropical cyclones reached Saffir–Simpson scale Category 5 intensity. North Atlantic Major Hurricane Michael’s landfall intensity of 140 kt was the fourth strongest for any continental U.S. hurricane landfall in the 168-year record. Michael caused more than 30 fatalities and $25 billion (U.S. dollars) in damages. In the western North Pacific, Super Typhoon Mangkhut led to 160 fatalities and$6 billion (U.S. dollars) in damages across the Philippines, Hong Kong, Macau, mainland China, Guam, and the Northern Mariana Islands. Tropical Storm Son-Tinh was responsible for 170 fatalities in Vietnam and Laos. Nearly all the islands of Micronesia experienced at least moderate impacts from various tropical cyclones. Across land, many areas around the globe received copious precipitation, notable at different time scales. Rodrigues and Réunion Island near southern Africa each reported their third wettest year on record. In Hawaii, 1262 mm precipitation at Waipā Gardens (Kauai) on 14–15 April set a new U.S. record for 24-h precipitation. In Brazil, the city of Belo Horizonte received nearly 75 mm of rain in just 20 minutes, nearly half its monthly average. Globally, fire activity during 2018 was the lowest since the start of the record in 1997, with a combined burned area of about 500 million hectares. This reinforced the long-term downward trend in fire emissions driven by changes in land use in frequently burning savannas. However, wildfires burned 3.5 million hectares across the United States, well above the 2000–10 average of 2.7 million hectares. Combined, U.S. wildfire damages for the 2017 and 2018 wildfire seasons exceeded $40 billion (U.S. dollars)

Model for Ub^ext affects on toxic protein aggregates.

<p>We propose that enhanced protein aggregate toxicity in Ub^ext-expressing cells is due to the inability of misfolded amyloidogenic proteins to be properly sequestered. The small soluble oligomers are more toxic than the large insoluble protein aggregates. UPS impairment caused by the expression of Ub^ext may hinder the rapid sequestration or retention of toxic oligomers into large protein aggregates.</p

Ub^ext expression increases cellular sensitivity to misfolded proteins.

<p>Ub^ext-expressing cells cannot tolerate excess misfolded proteins generated by the incorporation of canavanine. Serial dilutions of cells containing EV, Ub, or Ub^ext were spotted onto selective medium and selective medium containing 400 µM canavanine.</p

Expression of Ub^ext causes proteasomal impairment.

<p>(A) Ub^ext displays synthetic lethality with proteasome mutants. Wild type (WT) and temperature-sensitive proteasome mutant cells, <i>pre1-1 pre2-2</i> (11/22), were transformed with plasmids containing empty vector (EV), ubiquitin (Ub) and Ub^ext. Serial dilutions of cells were spotted onto selective medium and grown at 30°C and 37°C. (B) Cells expressing Ub^ext show a distinct pattern of ubiquitin conjugation. Protein lysate from wild type yeast cells containing an empty vector (WT), extra ubiquitin (Ub OE), or Ub^ext were analyzed by SDS-PAGE and western blot using an anti-ubiquitin antibody. Ub^ext causes an increase in ubiquitin-conjugated proteins (bracket) as compared to WT. The black arrowhead indicates ubiquitin monomer. The grey arrow points to Ub^ext. Black arrows represent conjugated Ub^ext. Pgk1p expression was probed to assess protein loading on the membrane. (C) Ub^ext-expressing cells impair the degradation of the N-end rule substrate R-β-galactosidase (βgal). Cells containing EV, Ub, or Ub^ext were transformed with pGal-Ub-R-<i>LacZ</i>. The stability of R-βgal was measured by specific activity (luminescence units/µg protein). The asterisk (*) indicates statistical significance between wild type Ub and Ub^ext (p = 0.0013). (D) Ub^ext-expression prevents the efficient proteasomal degradation of a ubiquitin fusion degradation substrate. The stability of Ub-P-<i>LacZ</i> was evaluated as in B. The asterisk (*) indicates statistical significance between wild type Ub and Ub^ext (p = 0.0005). (E) Ubiquitinated reporter substrates are present in Ub^ext-expressing cells. Wild type cells containing the Ub-X-<i>LacZ</i> reporter constructs and expressing Ub^ext or the control (EV) were analyzed for ubiquitinated βgal protein. βgal protein was immunoprecipitated with an anti-βgal antibody (left) and the bound fractions were blotted with an anti-ubiquitin antibody (right). The arrow indicates full length βgal protein.</p

Mutation of the Ub^ext hydrophobic patch (I44A) moderately affects proteasomal impairment.

<p>(A) Ub^extI44A still inhibits N-end rule substrate degradation. Cells containing pGal-Ub-R-<i>LacZ</i> were transformed with empty vector (EV), Ub, Ub^ext or Ub^extI44A (I44A) and the stability of R-βgal was measured by βgal activity assay. (B) Ub^extI44A moderately enhances the degradation of a UFD substrate. Cells containing pGal-Ub-P-<i>LacZ</i> were transformed with EV, Ub, Ub^ext or Ub^extI44A (I44A) and the stability of Ub-P-βgal was measured by βgal activity assay. The asterisk (*) indicates statistical significance between Ub^ext and Ub^extI44A (p = 0.0007).</p

Ub^ext alters the ubiquitination pattern of a UPS substrate.

<p>(A) R-βgal ubiquitination pattern is not altered in cells expressing Ub^ext. pGalUb-R-<i>LacZ</i> was transformed into proteasome mutant cells (<i>pre1-1 pre2-2</i>) expressing Ub^ext or EV and R-βgal was analyzed by immunoprecipitation (IP). Membranes were probed with anti-βgal and anti-ubiquitin antibodies. Arrow indicates full length βgal protein. (B) Ub-P-βgal ubiquitination is affected in cells expressing Ub^ext. Ub-P-βgal IPs were performed as in A. A subtle but reproducible difference in ubiquitination pattern was observed. Three independent IPs are shown. Arrowheads highlight distinct bands present in the EV lanes that are absent in Ub^ext lanes.</p

Expression of Ub^ext enhances the susceptibility of cells to toxic Sup35p aggregates but does not affect Sup35p aggregation.

<p>(A) [<i>PSI+</i>] cells expressing Ub^ext show reduced cell viability with lower induction of Sup35p. [<i>PSI+</i>] cells containing empty vector (EV), Ub^ext, UbΔGG, or Ub^extI44A were transformed with a copper-inducible <i>SUP35</i> or EV and analyzed for growth by spotting serial dilutions onto selective media containing 0, 50, or 100 µM CuSO₄. At 300 µM CuSO₄, [<i>PSI+</i>] cells over expressing Sup35p alone are not viable (not shown). (B) Prion conversion or induction was not enhanced in cells expressing Ub^ext. [<i>psi−</i>] cells expressing pSup35 or the control (EV) were transformed with empty vector (EV), Ub or Ub^ext and were analyzed for [<i>PSI+</i>] prion formation by monitoring colony color (the appearance of pink colonies). The graph represents the average of three independent cultures in which approximately 2,000 colonies per culture were evaluated for conversion. (C) Hsp104 protein levels are not enhanced in Ub^ext-expressing cells. Protein lysate from cells containing EV, Ub, or Ub^ext were subject to SDS-PAGE and western blot using an anti-Hsp104 antibody. Pgk1p expression was analyzed as a loading control. (D) The expression of Ub^ext did not alter cell survival in the presence of oxidative stress. Cells containing EV, Ub, or Ub^ext were treated with increasing concentrations of hydrogen peroxide (H₂O₂) and the number of viable cells was graphed as a percentage of the untreated. (E) The C-terminal domain of Sup35p (CTD) rescued the enhance susceptibility caused by Ub^ext in [<i>PSI+</i>] cells over expressing Sup35p. <i>Upper</i>: [<i>PSI+</i>]-mediated nonsense suppression is alleviated by expression of the CTD. [<i>PSI+</i>] cells containing EV show more nonsense suppression (the colony color is light pink). However, [<i>PSI+</i>] cells expressing the CTD display efficient translation termination and the colonies are red. <i>Lower</i>: [<i>PSI+</i>] cells expressing Ub^ext in addition to excess Sup35p (induced by 50 µM copper) are rescued from death by the expression of the CTD. (F) Sup35 protein aggregates were not altered by the presence of Ub^ext. Sup35p aggregates in strong [<i>PSI+</i>] ([<i>PSI+</i>]) and a weak strain variant of [<i>PSI+</i>] (w[<i>PSI+</i>]) were analyzed by SDD-AGE. The difference in Sup35p aggregate size of these prion strain variants can be appreciated by this method (compare [<i>PSI+</i>] to w[<i>PSI+</i>]). Sup35p aggregates from cells expressing excess Sup35p (OE Sup35p) and expressing Ub, Ub^ext, UbΔGG or containing an EV control were analyzed by SDD-AGE and western blot with an anti-Sup35 antibody.</p