Rab GTPases containing a CAAX motif are processed post-geranylgeranylation by proteolysis and methylation

Post-translational modification by protein prenylation is required for membrane targeting and biological function of monomeric GTPases. Ras and Rho proteins possess a C-terminal CAAX motif (C is cysteine, A is usually an aliphatic residue, and X is any amino acid), in which the cysteine is prenylated, followed by proteolytic cleavage of the AAX peptide and carboxyl methylation by the Rce1 CAAX protease and Icmt methyltransferase, respectively. Rab GTPases usually undergo double geranylgeranylation within CC or CXC motifs. However, very little is known about processing and membrane targeting of Rabs that naturally contain a CAAX motif. We show here that a variety of Rab-CAAX proteins undergo carboxyl methylation, both in vitro and in vivo, with one exception. Rab38(CAKS) is not methylated in vivo, presumably because of the inhibitory action of the lysine residue within the AAX motif for cleavage by Rce1. Unlike farnesylated Ras proteins, we observed no targeting defects of overexpressed Rab-CAAX proteins in cells deficient in Rce1 or Icmt, as reported for geranylgeranylated Rho proteins. However, endogenous geranylgeranylated non-methylated Rab-CAAX and Rab-CXC proteins were significantly redistributed to the cytosol at steady-state levels and redistribution correlates with higher affinity of RabGDI for non-methylated Rabs in Icmt-deficient cells. Our data suggest a role for methylation in Rab function by regulating the cycle of Rab membrane recruitment and retrieval. Our findings also imply that those Rabs that undergo post-prenylation processing follow an indirect targeting pathway requiring initial endoplasmic reticulum membrane association prior to specific organelle targeting.     

CTG trinucleotide repeat "big jumps": large expansions, small mice

Trinucleotide repeat expansions are the genetic cause of numerous human diseases, including fragile X mental retardation, Huntington disease, and myotonic dystrophy type 1. Disease severity and age of onset are critically linked to expansion size. Previous mouse models of repeat instability have not recreated large intergenerational expansions ("big jumps"), observed when the repeat is transmitted from one generation to the next, and have never attained the very large tract lengths possible in humans. Here, we describe dramatic intergenerational CTG*CAG repeat expansions of several hundred repeats in a transgenic mouse model of myotonic dystrophy type 1, resulting in increasingly severe phenotypic and molecular abnormalities. Homozygous mice carrying over 700 trinucleotide repeats on both alleles display severely reduced body size and splicing abnormalities, notably in the central nervous system. Our findings demonstrate that large intergenerational trinucleotide repeat expansions can be recreated in mice, and endorse the use of transgenic mouse models to refine our understanding of triplet repeat expansion and the resulting pathogenesis.     

Association between television in bedroom and adiposity throughout adolescence

Objective: The objective was to determine if having a television (TV) in the bedroom is associated with physical activity (PA), TV/video viewing, and adiposity throughout adolescence. Research Methods and Procedures: Longitudinal data (September 2002 through June 2005) were analyzed of 379 initially 12‐year‐old French adolescents participating as controls in the Intervention Centered on Adolescents’ Physical activity and Sedentary behavior (ICAPS). Presence of a TV set in the bedroom (TV_bedroom) and leisure activities were obtained by questionnaire. There was annual assessment of BMI, waist circumference, and body fat by bioimpedance. Results: In boys but not girls, baseline TV_bedroom was associated with higher TV/video viewing over time [odds ratio (OR) of high TV/video = 1.87; 95% confidence interval, 1.2 to 2.8] and less no‐sport club participation (OR = 0.59; 95% confidence interval, 0.35 to 1.0). Both boys and girls with baseline TV_bedroom had lower reading time (p < 0.0001 in boys; p = 0.04 in girls), while PA did not differ according to TV_bedroom for boys or for girls. For boys only, baseline TV_bedroom was associated with higher BMI (mean BMI over time 20.5 ± 0.5 vs. 19.0 ± 0.5 kg/m²; p = 0.001), waist circumference (70.9 ± 0.9 vs. 67.2 ± 0.8 cm; p < 0.001), and body fat (15.9 ± 0.9% vs. 13.5 ± 0.9%; p < 0.001), without interaction with time. These relationships remained significant after adjustment for socioeconomic status. TV/video viewing explained 26%, 42%, and 36% of the relationships of TV_bedroom with BMI, waist circumference, and body fat, respectively, while addition of other leisure activities in the models only marginally reduced the effects. Discussion: These results suggest the importance of keeping TV out of an adolescent's bedroom from an obesity prevention perspective but show gender differences.     

Prolonged starvation drives reversible sequestration of lipid biosynthetic enzymes and organelle reorganization in Saccharomyces cerevisiae

Cells adapt to changing nutrient availability by modulating a variety of processes, including the spatial sequestration of enzymes, the physiological significance of which remains controversial. These enzyme deposits are claimed to represent aggregates of misfolded proteins, protein storage, or complexes with superior enzymatic activity. We monitored spatial distribution of lipid biosynthetic enzymes upon glucose depletion in Saccharomyces cerevisiae. Several different cytosolic-, endoplasmic reticulum–, and mitochondria-localized lipid biosynthetic enzymes sequester into distinct foci. Using the key enzyme fatty acid synthetase (FAS) as a model, we show that FAS foci represent active enzyme assemblies. Upon starvation, phospholipid synthesis remains active, although with some alterations, implying that other foci-forming lipid biosynthetic enzymes might retain activity as well. Thus sequestration may restrict enzymes'' access to one another and their substrates, modulating metabolic flux. Enzyme sequestrations coincide with reversible drastic mitochondrial reorganization and concomitant loss of endoplasmic reticulum–mitochondria encounter structures and vacuole and mitochondria patch organelle contact sites that are reflected in qualitative and quantitative changes in phospholipid profiles. This highlights a novel mechanism that regulates lipid homeostasis without profoundly affecting the activity status of involved enzymes such that, upon entry into favorable growth conditions, cells can quickly alter lipid flux by relocalizing their enzymes.     

Reactive oxygen species are involved in eosinophil extracellular traps release and in airway inflammation in asthma

In asthma, there are high levels of inflammatory mediators, reactive oxygen species (ROS), and eosinophil extracellular traps (EETs) formation in airway. Here, we attempted to investigate the ROS involvement in EETs release and airway inflammation in OVA-challenged mice. Before the intranasal challenge with ovalbumin (OVA), animals were treated with two ROS inhibitors, N-acetylcysteine (NAC) or diphenyleneiodonium (DPI). We showed that NAC treatment reduced inflammatory cells in lung. DPI and NAC treatments reduced eosinophil peroxidase (EPO), goblet cells hyperplasia, proinflammatory cytokines, NFκB p65 immunocontent, and oxidative stress in lung. However, only the NAC treatment improved mitochondrial energy metabolism. Moreover, the treatments with DPI and NAC reduced EETs release in airway. This is the first study to show that ROS are needed for EETs formation in asthma. Based on our results, NAC and DPI treatments can be an interesting alternative for reducing airway inflammation, mitochondrial damage, and EETs release in asthma.     

Impact of cell culture media additives on IgG glycosylation produced in Chinese hamster ovary cells

Glycosylation is a key critical quality attribute for monoclonal antibodies and other recombinant proteins because of its impact on effector mechanisms and half-life. In this study, a variety of compounds were evaluated for their ability to modulate glycosylation profiles of recombinant monoclonal antibodies produced in Chinese hamster ovary cells. Compounds were supplemented into the cell culture feed of fed-batch experiments performed with a CHO K1 and a CHO DG44 cell line expressing a recombinant immunoglobulin G1 (IgG1). Experiments were performed in spin tubes or the ambr®15 controlled bioreactor system, and the impact of the compounds at various concentrations was determined by monitoring the glycosylation profile of the IgG and cell culture parameters, such as viable cell density, viability, and titer. Results indicate that the highest impact on mannosylation was achieved through 15 µM kifunensine supplementation leading to an 85.8% increase in high-mannose containing species. Fucosylation was reduced by 76.1% through addition of 800 µM 2-F-peracetyl fucose. An increase of 40.9% in galactosylated species was achieved through the addition of 120 mM galactose in combination with 48 µM manganese and 24 µM uridine. Furthermore, 6.9% increased sialylation was detected through the addition of 30 µM dexamethasone in combination with the same manganese, uridine, and galactose mixture used to increase total galactosylation. Further compounds or combinations of additives were also efficient at achieving a smaller overall glycosylation modulation, required, for instance, during the development of biosimilars. To the best of our knowledge, no evaluation of the efficacy of such a variety of compounds in the same cell culture system has been described. The studied cell culture media additives are efficient modulators of glycosylation and are thus a valuable tool to produce recombinant glycoproteins.     

Silencing the OCT4-PG1 pseudogene reduces OCT-4 protein levels and changes characteristics of the multidrug resistance phenotype in chronic myeloid leukemia

Molecular Biology Reports - Cancer stem cells show epigenetic plasticity and intrinsic resistance to anti-cancer therapy, rendering capable of initiating cancer relapse and progression....