A Combined Approach of Quantitative Interaction Proteomics and Live-cell Imaging Reveals a Regulatory Role for Endoplasmic Reticulum (ER) Reticulon Homology Proteins in Peroxisome Biogenesis

Peroxisome biogenesis initiates at the endoplasmic reticulum (ER) and maturation allows for the formation of metabolically active organelles. Yet, peroxisomes can also multiply by growth and division. Several proteins, called peroxins, are known to participate in these processes but little is known about their organization to orchestrate peroxisome proliferation. Here, we demonstrate that regulation of peroxisome proliferation relies on the integrity of the tubular ER network. Using a dual track SILAC-based quantitative interaction proteomics approach, we established a comprehensive network of stable as well as transient interactions of the peroxin Pex30p, an integral membrane protein. Through association with merely ER resident proteins, in particular with proteins containing a reticulon homology domain, and with other peroxins, Pex30p designates peroxisome contact sites at ER subdomains. We show that Pex30p traffics through the ER and segregates in punctae to which peroxisomes specifically append, and we ascertain its transient interaction with all subunits of the COPI coatomer complex suggesting the involvement of a vesicle-mediated transport. We establish that the membrane protein Pex30p facilitates the connection of peroxisomes to the ER. Taken together, our data indicate that Pex30p-containing protein complexes act as focal points from which peroxisomes can form and that the tubular ER architecture organized by the reticulon homology proteins Rtn1p, Rtn2p and Yop1p controls this process.All nucleated cells contain essential round-shaped organelles called peroxisomes, whose function is mainly associated with lipid metabolism (1). Depending on the cellular requirements, the size, number, and protein content of these single membrane-bound organelles can vary widely. Although peroxisomes are dispensable for unicellular species such as yeasts, they are essential for the development of multicellular organisms (2, 3). In human, mutations in PEX genes lead to defects in peroxisome function or formation and are associated with the development of lethal pathologies (4). These PEX genes code for proteins, called peroxins, which are involved in peroxisome assembly and maintenance (5).Two major routes seem to lead to peroxisome formation, namely, de novo biogenesis and growth/division of pre-existing peroxisomes. The division pathway operates with proteins of the Pex11 family and requires fission factors shared with mitochondria (6). Studies in yeast and mammalian cells revealed that through the action of the protein Pex3p peroxisome precursors can also originate from the endoplasmic reticulum (ER)¹ and, via import of membrane and matrix proteins, mature into fully functional organelles (7, 8). Furthermore, several peroxisomal membrane proteins were shown to migrate to peroxisomes via the ER (7, 9, 10). The molecular mechanism underlying the biogenic pathway of peroxisome formation has not been clarified so far. Recent data based on cell-free vesicle-budding reactions, however, demonstrated that several peroxisomal proteins traffic from the ER to peroxisomes in a COPII vesicle-independent manner (11). These observations point to the existence of vesicular events to mediate the transport of peroxisomal membrane proteins from the ER. In fact, analysis of secretory mutant yeast cells already suggest that part of the ER-associated secretory machinery is involved in peroxisome biogenesis (12).The de novo biogenesis of peroxisomes and the growth/division pathways are usually seen as independent routes; however, these events may be coordinated and, thus, intimately linked. Indeed, peroxisomes need to acquire membrane components to proliferate and it has been proposed that their binding to the cell cortex or to the cytoskeleton allows their partitioning and segregation during cell division (13–15).Among the proteins required for assembly of peroxisomes, the membrane proteins Pex23p and Pex24p play essential roles in the yeast Yarrowia lipolytica (16, 17). Homologs of these two proteins in Saccharomyces cerevisiae are Pex30p, Pex31p, and Pex32p, all containing at least one transmembrane domain and a dysferlin domain as common structural motifs, as well as Pex28p and Pex29p. In S. cerevisiae, these proteins seem to negatively control peroxisomal size and number (18, 19). Interestingly, Pex30p seems to exhibit species-specific differences in the regulation of peroxisome proliferation. While the lack of Pex30p in S. cerevisiae leads to an increase in the number of normal-sized peroxisomes (18), in Pichia pastoris its absence correlates with the appearance of fewer and clustered peroxisomes (20). Although peroxisomes are highly versatile organelles, under given conditions their total number per cell remains fairly constant owing to the delicate balance of proliferation, inheritance and degradation (21, 22). The question is: what are the molecular mechanisms responsible for the spatiotemporal organization of these events?Here, we present data obtained from a dual approach based on quantitative interaction proteomics using stable isotope labeling with amino acids in cell culture (SILAC) (23, 24) and live-cell imaging, revealing for the first time the dynamic interaction network around Pex30p and its function in the organization of ER-to-peroxisome membrane associations. We report the existence of a macromolecular membrane protein complex that acts as a hub for the regulation of peroxisome proliferation and movement. Our data suggest a direct role for the tubular cortical ER and the reticulon homology proteins Rtn1p, Rtn2p, and Yop1p in the regulation of peroxisome biogenesis. Furthermore, as an initially cortical-ER localized protein that interacts with reticulon homology proteins, Pex30p is shown in this work to establish contacts between ER tubules and peroxisomes and to specifically traffic through the ER. In summary, our data reveal a central role for Pex30p in the formation of ER-to-peroxisomes associations that appear to be involved in the coordination of peroxisome biogenesis and maintenance.     

Magnetic Resonance Colonography for Fibrosis Assessment in Rats with Chronic Colitis

Background

Magnetic resonance colonography (MRC) has been developed to assess inflammatory bowel diseases. We aimed to assess the feasibility of MRC in rats with TNBS-induced chronic colitis and to confront imaging results with fibrosis and stenosing features of the model.

Materials and Methods

Chronic colitis was induced in 12 rats by weekly intra-rectal injection of increasing doses of TNBS for 6 weeks, while 8 control rats received the vehicle. At week 7, MRC was performed. Fibrosis scores were assessed and fibrosis mediators measured.

Results

Chronic colitis was associated with significant body weight loss (p<0.0001) and higher colon weight/length compared to controls (p = 0.0004). Fibrosis mediators and histological scores were significantly higher in rats with TNBS than in controls: α-SMA expression (0.9 versus 0.61, p = 0.0311) and fibrosis score (p = 0.0308). Colon wall thickness was higher in rats with TNBS than in controls: maximal thickness (2.38 versus 0.74 mm, p<0.0001) and minimal thickness (1.33 versus 0.48 mm, p<0.0001). Wall signal intensity on T2w images was higher in rats with TNBS than in controls (9040 versus 6192, p = 0.0101) and correlated with fibrosis score (r = 0.5214; p = 0.04). Luminal narrowing was higher in rats with TNBS (50.08 versus 10.33%, p<0.0001) and correlated with α-SMA expression (r = 0.5618; p = 0.01). Stenosis was observed in 7/9 rats with TNBS and in no controls (p = 0.0053).

Conclusions

MRC is feasible and easily distinguishes rats with colitis from controls. MRC signs correlated with fibrosis parameters. MRC evaluation may be part of a new anti-fibrosis drug assessment in experimental models of chronic colitis.     

Negative Events in Childhood Predict Trajectories of Internalizing Symptoms Up to Young Adulthood: An 18-Year Longitudinal Study

Background

Common negative events can precipitate the onset of internalizing symptoms. We studied whether their occurrence in childhood is associated with mental health trajectories over the course of development.

Methods

Using data from the TEMPO study, a French community-based cohort study of youths, we studied the association between negative events in 1991 (when participants were aged 4–16 years) and internalizing symptoms, assessed by the ASEBA family of instruments in 1991, 1999, and 2009 (n = 1503). Participants'' trajectories of internalizing symptoms were estimated with semi-parametric regression methods (PROC TRAJ). Data were analyzed using multinomial regression models controlled for participants'' sex, age, parental family status, socio-economic position, and parental history of depression.

Results

Negative childhood events were associated with an increased likelihood of concurrent internalizing symptoms which sometimes persisted into adulthood (multivariate ORs associated with > = 3 negative events respectively: high and decreasing internalizing symptoms: 5.54, 95% CI: 3.20–9.58; persistently high internalizing symptoms: 8.94, 95% CI: 2.82–28.31). Specific negative events most strongly associated with youths'' persistent internalizing symptoms included: school difficulties (multivariate OR: 5.31, 95% CI: 2.24–12.59), parental stress (multivariate OR: 4.69, 95% CI: 2.02–10.87), serious illness/health problems (multivariate OR: 4.13, 95% CI: 1.76–9.70), and social isolation (multivariate OR: 2.24, 95% CI: 1.00–5.08).

Conclusions

Common negative events can contribute to the onset of children''s lasting psychological difficulties.     

Incidence of Cyp51 A Key Mutations in Aspergillus fumigatus—A Study on Primary Clinical Samples of Immunocompromised Patients in the Period of 1995–2013

As the incidence of azole resistance in Aspergillus fumigatus is rising and the diagnosis of invasive aspergillosis (IA) in immunocompromised patients is rarely based on positive culture yield, we screened our Aspergillus DNA sample collection for the occurrence of azole resistance mediating cyp51 A key mutations. Using two established, a modified and a novel polymerase chain reaction (PCR) assays followed by DNA sequence analysis to detect the most frequent mutations in the A. fumigatus cyp51 A gene conferring azole resistance (TR34 (tandem repeat), L98H and M220 alterations). We analyzed two itraconazole and voriconazole and two multi-azole resistant clinical isolates and screened 181 DNA aliquots derived from clinical samples (blood, bronchoalveolar lavage (BAL), biopsies, cerebrospinal fluid (CSF)) of 155 immunocompromised patients of our Aspergillus DNA sample collection, previously tested positive for Aspergillus DNA and collected between 1995 and 2013. Using a novel PCR assay for the detection of the cyp51 A 46 bp tandem repeat (TR46) directly from clinical samples, we found the alteration in a TR46/Y121F/T289A positive clinical isolate. Fifty stored DNA aliquots from clinical samples were TR46 negative. DNA sequence analysis revealed a single L98H mutation in 2010, two times the L98H alteration combined with TR34 in 2011 and 2012 and a so far unknown N90K mutation in 1998. In addition, four clinical isolates were tested positive for the TR34/L98H combination in the year 2012. We consider our assay of epidemiological relevance to detect A. fumigatus azole resistance in culture-negative clinical samples of immunocompromised patients; a prospective study is ongoing.     

Selective attenuation of norepinephrine release and stress-induced heart rate increase by partial adenosine A1 agonism

The release of the neurotransmitter norepinephrine (NE) is modulated by presynaptic adenosine receptors. In the present study we investigated the effect of a partial activation of this feedback mechanism. We hypothesized that partial agonism would have differential effects on NE release in isolated hearts as well as on heart rate in vivo depending on the genetic background and baseline sympathetic activity. In isolated perfused hearts of Wistar and Spontaneously Hypertensive Rats (SHR), NE release was induced by electrical stimulation under control conditions (S1), and with capadenoson 6 · 10(-8) M (30 μg/l), 6 · 10(-7) M (300 μg/l) or 2-chloro-N(6)-cyclopentyladenosine (CCPA) 10(-6) M (S2). Under control conditions (S1), NE release was significantly higher in SHR hearts compared to Wistar (766+/-87 pmol/g vs. 173+/-18 pmol/g, p<0.01). Capadenoson led to a concentration-dependent decrease of the stimulation-induced NE release in SHR (S2/S1 = 0.90 ± 0.08 with capadenoson 6 · 10(-8) M, 0.54 ± 0.02 with 6 · 10(-7) M), but not in Wistar hearts (S2/S1 = 1.05 ± 0.12 with 6 · 10(-8) M, 1.03 ± 0.09 with 6 · 10(-7) M). CCPA reduced NE release to a similar degree in hearts from both strains. In vivo capadenoson did not alter resting heart rate in Wistar rats or SHR. Restraint stress induced a significantly greater increase of heart rate in SHR than in Wistar rats. Capadenoson blunted this stress-induced tachycardia by 45% in SHR, but not in Wistar rats. Using a [(35)S]GTPγS assay we demonstrated that capadenoson is a partial agonist compared to the full agonist CCPA (74+/-2% A(1)-receptor stimulation). These results suggest that partial adenosine A(1)-agonism dampens stress-induced tachycardia selectively in rats susceptible to strong increases in sympathetic activity, most likely due to a presynaptic attenuation of NE release.     

CD4 dynamics over a 15 year-period among HIV controllers enrolled in the ANRS French observatory

Background

There are few large published studies of HIV controllers with long-term undetectable viral load (VL). We describe the characteristics and outcomes of 81 French HIV controllers.

Methods and Results

HIV controllers were defined as asymptomatic, antiretroviral-naïve persons infected ≥10 years previously, with HIV-RNA <400 copies/mL in >90% of plasma samples. All available CD4 and VL values were collected at enrolment. Mixed-effect linear models were used to analyze CD4 cell count slopes since diagnosis. HIV controllers represented 0.31% of all patients managed in French hospitals. Patients infected through intravenous drug use were overrepresented (31%) and homosexual men were underrepresented (26% of men) relative to the ANRS SEROCO cohort of subjects diagnosed during the same period. HIV controllers whose VL values were always below the detection limit of the assays were compared with those who had rare “blips” (<50% of VL values above the detection limit) or frequent blips (>50% of VL values above the detection limit). Estimated CD4 cell counts at HIV diagnosis were similar in the three groups. CD4 cell counts remained stable after HIV diagnosis in the “no blip” group, while they fell significantly in the two other groups (−0.26√CD4 and −0.28√CD4/mm³/year in the rare and frequent blip groups, respectively). No clinical, immunological or virological progression was observed in the no blip group, while 3 immunological and/or virological events and 4 cancers were observed in the blip subgroups.

Conclusions

Viral blips in HIV controllers are associated with a significant decline in CD4 T cells and may be associated with an increased risk of pathological events, possibly owing to chronic inflammation/immune activation.     

Syndecan-syntenin-ALIX regulates the biogenesis of exosomes

The biogenesis of exosomes, small secreted vesicles involved in signalling processes, remains incompletely understood. Here, we report evidence that the syndecan heparan sulphate proteoglycans and their cytoplasmic adaptor syntenin control the formation of exosomes. Syntenin interacts directly with ALIX through LYPX(n)L motifs, similarly to retroviral proteins, and supports the intraluminal budding of endosomal membranes. Syntenin exosomes depend on the availability of heparan sulphate, syndecans, ALIX and ESCRTs, and impact on the trafficking and confinement of FGF signals. This study identifies a key role for syndecan-syntenin-ALIX in membrane transport and signalling processes.     

Food Insecurity and Children’s Mental Health: A Prospective Birth Cohort Study

Food insecurity (which can be defined as inadequate access to sufficient, safe, and nutritious food that meets individuals’ dietary needs) is concurrently associated with children’s psychological difficulties. However, the predictive role of food insecurity with regard to specific types of children’s mental health symptoms has not previously been studied. We used data from the Longitudinal Study of Child Development in Québec, LSCDQ, a representative birth cohort study of children born in the Québec region, in Canada, in 1997–1998 (n = 2120). Family food insecurity was ascertained when children were 1½ and 4½ years old. Children’s mental health symptoms were assessed longitudinally using validated measures of behaviour at ages 4½, 5, 6 and 8 years. Symptom trajectory groups were estimated to identify children with persistently high levels of depression/anxiety (21.0%), aggression (26.2%), and hyperactivity/inattention (6.0%). The prevalence of food insecurity in the study was 5.9%. In sex-adjusted analyses, children from food-insecure families were disproportionately likely to experience persistent symptoms of depression/anxiety (OR: 1.79, 95% CI 1.15–2.79) and hyperactivity/inattention (OR: 3.06, 95% CI 1.68–5.55). After controlling for immigrant status, family structure, maternal age at child’s birth, family income, maternal and paternal education, prenatal tobacco exposure, maternal and paternal depression and negative parenting, only persistent hyperactivity/inattention remained associated with food insecurity (fully adjusted OR: 2.65, 95% CI 1.16–6.06). Family food insecurity predicts high levels of children’s mental health symptoms, particularly hyperactivity/inattention. Addressing food insecurity and associated problems in families could help reduce the burden of mental health problems in children and reduce social inequalities in development.     

Cyclophilin B induces integrin-mediated cell adhesion by a mechanism involving CD98-dependent activation of protein kinase C-delta and p44/42 mitogen-activated protein kinases

Initially identified as a cyclosporin-A binding protein, cyclophilin B (CyPB) is an inflammatory mediator that induces adhesion of T lymphocytes to fibronectin, by a mechanism dependent on CD147 and alpha 4 beta 1 integrins. Recent findings have suggested that another cell membrane protein, CD98, may cooperate with CD147 to regulate beta1 integrin functions. Based on these functional relationships, we examined the contribution of CD98 in the pro-adhesive activity of CyPB, by utilizing the responsive promonocyte cell line THP-1. We demonstrated that cross-linking CD98 with CD98-AHN-18 antibody mimicked the responses induced by CyPB, i.e. homotypic aggregation, integrin-mediated adhesion to fibronectin and activation of p44/42 MAPK. Consistent with previous data, immunoprecipitation confirmed the existence of a heterocomplex wherein CD147, CD98 and beta1 integrins were associated. We then demonstrated that CyPB-induced cell adhesion and p44/42 MAPK activation were dependent on the participation of phosphoinositide 3-kinase and subsequent activation of protein kinase C-delta. Finally, silencing the expression of CD98 by RNA interference potently reduced CyPB-induced cell responses, thus confirming the role of CD98 in the pro-adhesive activity of CyPB. Altogether, our results support a model whereby CyPB induces integrin-mediated adhesion via interaction with a multimolecular unit formed by the association between CD147, CD98 and beta1 integrins.