Ubiquitin-conjugating enzymes UBC4 and UBC5 mediate selective degradation of short-lived and abnormal proteins.

Ubiquitin-conjugating enzymes catalyse the covalent attachment of ubiquitin to target proteins. Members of this enzyme family are involved in strikingly diverse cellular functions: UBC2 (RAD6) is central to DNA repair, UBC3 (CDC34) is involved in cell cycle control. We have cloned the genes for two novel ubiquitin-conjugating enzymes, UBC4 and UBC5, from the yeast Saccharomyces cerevisiae. These enzymes mediate selective degradation of short-lived and abnormal proteins. UBC4 and UBC5 are closely related in sequence and complementing in function. Expression of UBC4 and UBC5 genes is heat inducible. UBC4 and UBC5 enzymes generate high mol. wt ubiquitin-protein conjugates in vivo consistent with previous studies which suggested that attachment of multiple ubiquitin molecules to proteolytic substrates is required for their selective degradation. UBC4 and UBC5 enzymes comprise a major part of total ubiquitin-conjugation activity in stressed cells. Turnover of short-lived proteins and canavanyl-peptides but not of long-lived proteins is markedly reduced in ubc4ubc5 mutants. Loss of UBC4 and UBC5 activity impairs cell growth, leads to inviability at elevated temperatures or in the presence of an amino acid analog, and induces the stress response.     

The conserved ubiquitin-like protein Hub1 plays a critical role in splicing in human cells

Different from canonical ubiquitin-like proteins, Hub1 does not form covalent conjugates with substrates but binds proteins noncovalently. In Socchoromyces cerevisioe, Hub1 associates with spUceosomes and mediates alternative splicing of SRCI, without affecting pre-mRNA splicing generaity. Human Hub1 is highty similar to its yeast homotog, but its cellular function remains largely unexplored. Here, we show that human Hub1 binds to the spliceosomal protein Snu66 as in yeast; however, unlike its 5. cerevisioe homolos, human Hub1 is essential for viability. Prolonged in vivo depletion of human Hub1 leads to various cellular defects, including splicing speckle abnormalities, partial nuclear retention of mRNAs, mitotic catastrophe, and consequently cell death by apoptosis. Early consequences of Hub1 depletion are severe splicing defects, however, only for specific splice sites leading to exon skipping and intron retention. Thus, the ubiquitin-iike protein Hub1 is not a canonlcal spliceosomal factor needed generally for splicing, but rather a modulator of spliceosome performance and facilitator of alternative splicing.     

Noncanonical Role of the 9-1-1 Clamp in the Error-Free DNA Damage Tolerance Pathway

1. Download : Download high-res image (310KB)
2. Download : Download full-size image

Highlights? 9-1-1 and Exo1 are components of the error-free RAD6 pathway ? 9-1-1 promotes postreplicative template switching ? Polyubiquitylated PCNA and 9-1-1 cooperate in the error-free RAD6 pathway ? 9-1-1’s role in the error-free RAD6 pathway is uncoupled from checkpoint functions     

In-depth qualitative and quantitative analysis of composite glycosylation profiles and other micro-heterogeneity on intact monoclonal antibodies by high-resolution native mass spectrometry using a modified Orbitrap

Here, we describe a fast, easy-to-use, and sensitive method to profile in-depth structural micro-heterogeneity, including intricate N-glycosylation profiles, of monoclonal antibodies at the native intact protein level by means of mass spectrometry using a recently introduced modified Orbitrap Exactive Plus mass spectrometer. We demonstrate the versatility of our method to probe structural micro-heterogeneity by describing the analysis of three types of molecules: (1) a non-covalently bound IgG4 hinge deleted full-antibody in equilibrium with its half-antibody, (2) IgG4 mutants exhibiting highly complex glycosylation profiles, and (3) antibody-drug conjugates. Using the modified instrument, we obtain baseline separation and accurate mass determination of all different proteoforms that may be induced, for example, by glycosylation, drug loading and partial peptide backbone-truncation. We show that our method can handle highly complex glycosylation profiles, identifying more than 20 different glycoforms per monoclonal antibody preparation and more than 30 proteoforms on a single highly purified antibody. In analyzing antibody-drug conjugates, our method also easily identifies and quantifies more than 15 structurally different proteoforms that may result from the collective differences in drug loading and glycosylation. The method presented here will aid in the comprehensive analytical and functional characterization of protein micro-heterogeneity, which is crucial for successful development and manufacturing of therapeutic antibodies     

Patterns of island treeline elevation – a global perspective

Treeline research has strongly focused on mountain systems on the mainland. However, island treelines offer the opportunity to contribute to the global framework on treeline elevation due to their island‐specific attributes such as isolation, small area, low species richness and relative youth. We hypothesize that, similar to the mainland, latitude‐driven temperature variation is the most important determinant of island treeline elevation on a global scale. To test this hypothesis, we compared mainland with island treeline elevations. Then we focused 1) on the global effects of latitude, 2) on the regional effects of island type (continental vs oceanic islands) and 3) the local effects of several specific island characteristics (age, area, maximum island elevation, isolation and plant species richness). We collected a global dataset of islands (n = 86) by applying a stratified design using GoogleEarth and the Global Island Database. For each island we extracted data on latitude and local characteristics. Treeline elevation decreased from the mainland through continental to oceanic islands. Island treeline elevation followed a hump‐shaped latitudinal distribution, which is fundamentally different from the mainland double‐hump. Higher maximum island elevation generated higher treeline elevation and was found the best single predictor of island treeline elevation, even better than latitude. Lower island treeline elevation may be the result of a low mass elevation effect (MEE) influencing island climates and an increasingly impoverished species pool but also trade wind inversion‐associated aridity. The maximum island elevation effect possibly results from an increasing mass elevation effect (MEE) with increasing island elevation but also range shifts during climatic fluctuations and the summit syndrome (i.e. high wind speeds and poor soils in peak regions). Investigating islands in treeline research has enabled disentangling the global effect of latitude from regional and local effects and, at least for islands, a comprehensive quantification of the MEE.     

Methane production in cattle calculated by the nutrient composition of the diet

In this study data originating from complete metabolic trials with cattle of both sexes, fed 337 rations at feed intake levels between one to three times maintenance energy requirement were used to regress the total CH4 emission to the level of DM intake and to the nutrient composition, respectively. A major component of the measured CH4 emission cannot be explained by DM intake but is rather due to differences in dietary nutrient composition. The amount of digestible nutrients especially of the carbohydrate fraction (starch, sugar, N-free residuals) are reliable to estimate CH4 release with high precision (r2 = 0.885). Its production rate increased to 1.9-fold higher levels (range 1.8-2.1) per g of N-free residuals compared to that induced per g of protein, starch or sugar, respectively. Furthermore, diets rich in fat reduced CH4 formation in the rumen. The regression equations fit a wide range of diets and diet compositions, and more importantly, they are applicable to various types of production systems.     

Facilitating haplotype analysis by fully automated analysis of all chromosomes in human-mouse hybrid cell lines

Recent evidence suggests that haplotype analysis is essential in recognizing genetic factors involved in the tendency toward a particular disease or pharmacogenetic phenotype, as well as to identify genes involved in multigenic disorders. Because of the increasing need for efficient haplotype tests, a new hybrid system, called conversion technology, was developed. Conversion technology aims at converting the diploid chromosome content into a haploid state so that hybrids contain a single copy of any desired chromosome. A number of mutations can now be identified easily, as they are no longer obscured by the normal sequence present on the other copy of the chromosome. However, the efficient use of this hybrid system depends on a complete analysis of both human and mouse chromosome complements in order to assess the stability of the hybrid cells and to accurately determine their human chromosome content. We describe a new multicolor FISH-based method capable of analyzing both genomes simultaneously in a single hybridization. This new technique should become an instrumental part of inexpensive, reliable haplotype tests.     

Effect of a soy protein diet on protein and energy metabolism and organ development in protein-restricted growing pigs

Two feeding experiments were carried out with castrated male pigs weighing between 10 and 30 kg to study acute and persisting dietary effects on growth and on protein and energy metabolism in growing pigs. Pigs were fed semi-synthetic isoenergetic and isonitrogenous diets at 50% protein requirement with either soy protein isolate (SPI) or casein (CAS) as sole protein source. Intake of protein and ME amounted to 9% w/w and 1800 kJ · kg BW ^{? 0.62} · d ^{? 1} in Exp. 1, respectively, and 9% w/w and 1430 kJ · kg BW ^{? 0.62} · d ^{? 1} in Exp. 2. The CAS diet was supplemented by Lys, Met, Thr and Trp. In Exp. 1 (acute effects), 18 pigs received the CAS diet for 24 days (period 1); 9 pigs were then switched to a SPI diet whereas 9 pigs continued on the CAS diet for another 31 days (period 2). In Exp. 2, a third period of 31 days was added in which the SPI group was switched back to CAS diet. The control group was fed on the CAS diet throughout Exp. 2 (86 days). Altogether the majority of parameters were not affected neither comparing SPI with CAS in Exp. 1 nor inspecting possible persistence of effects in Exp. 2. In detail, in Exp. 1 SPI compared to CAS feeding resulted in a lower efficiency of protein utilisation and lower protein retention. Attendant upon the lower protein retention an increased energy retention as fat was only observed in tendency. SPI feeding caused a decreased body weight, thyroid weight and increased hepatic carbohydrate content that persisted after the diet was changed back to CAS (Exp. 2).     

Plant responses to climatic extremes: within‐species variation equals among‐species variation

Within‐species and among‐species differences in growth responses to a changing climate have been well documented, yet the relative magnitude of within‐species vs. among‐species variation has remained largely unexplored. This missing comparison impedes our ability to make general predictions of biodiversity change and to project future species distributions using models. We present a direct comparison of among‐ versus within‐species variation in response to three of the main stresses anticipated with climate change: drought, warming, and frost. Two earlier experiments had experimentally induced (i) summer drought and (ii) spring frost for four common European grass species and their ecotypes from across Europe. To supplement existing data, a third experiment was carried out, to compare variation among species from different functional groups to within‐species variation. Here, we simulated (iii) winter warming plus frost for four grasses, two nonleguminous, and two leguminous forbs, in addition to eleven European ecotypes of the widespread grass Arrhenatherum elatius. For each experiment, we measured: (i) C/N ratio and biomass, (ii) chlorophyll content and biomass, and (iii) plant greenness, root ¹⁵N uptake, and live and dead tissue mass. Using coefficients of variation (CVs) for each experiment and response parameter, a total of 156 within‐ vs. among‐species comparisons were conducted, comparing within‐species variation in each of four species with among‐species variation for each seed origin (five countries). Of the six significant differences, within‐species CVs were higher than among‐species CVs in four cases. Partitioning of variance within each treatment in two of the three experiments showed that within‐species variability (ecotypes) could explain an additional 9% of response variation after accounting for the among‐species variation. Our observation that within‐species variation was generally as high as among‐species variation emphasizes the importance of including both within‐ and among‐species variability in ecological theory (e.g., the insurance hypothesis) and for practical applications (e.g., biodiversity conservation).