Self-interaction of human Pex11pβ during peroxisomal growth and division.

Journal ArticleResearch Support, Non-U.S. Gov'tCopyright: © 2013 Bonekamp et al.Pex11 proteins are involved in membrane elongation and division processes associated with the multiplication of peroxisomes. Human Pex11pβ has recently been linked to a new disorder affecting peroxisome morphology and dynamics. Here, we have analyzed the exact membrane topology of Pex11pβ. Studies with an epitope-specific antibody and protease protection assays show that Pex11pβ is an integral membrane protein with two transmembrane domains flanking an internal region exposed to the peroxisomal matrix and N- and C-termini facing the cytosol. A glycine-rich internal region within Pex11pβ is dispensable for peroxisome membrane elongation and division. However, we demonstrate that an amphipathic helix (Helix 2) within the first N-terminal 40 amino acids is crucial for membrane elongation and self-interaction of Pex11pβ. Interestingly, we find that Pex11pβ self-interaction strongly depends on the detergent used for solubilization. We also show that N-terminal cysteines are not essential for membrane elongation, and that putative N-terminal phosphorylation sites are dispensable for Pex11pβ function. We propose that self-interaction of Pex11pβ regulates its membrane deforming activity in conjunction with membrane lipids.Portuguese Foundation for Science and Technology (FCT)FEDERCRUP/DAA

Predicting the targeting of tail-anchored proteins to subcellular compartments in mammalian cells

This is the author accepted manuscript. The final version is available from Company of Biologists via the DOI in this record.Tail-anchored (TA) proteins contain a single transmembrane domain (TMD) at the Cterminus, anchoring them to organelle membranes where they mediate a variety of critical cellular processes. Mutations in individual TA proteins cause a number of severe inherited disorders. However, the molecular mechanisms and signals facilitating proper TA protein targeting are not fully understood, in particular in mammals. Here, we identify additional TA proteins at peroxisomes or shared by multiple organelles in mammals and reveal that a combination of TMD hydrophobicity and tail charge determines targeting to distinct organelles. Specifically, an increase in tail charge can override a hydrophobic TMD signal and re-direct a protein from the ER to peroxisomes or mitochondria and vice versa. We demonstrate that subtle alterations in those physicochemical parameters can shift TA protein targeting between organelles, explaining why peroxisomes and mitochondria share many TA proteins. Our analyses enabled us to allocate characteristic physicochemical parameters to different organelle groups. This classification allows for the first time, successful prediction of the location of uncharacterized TA proteins.We thank colleagues who provided materials (see Tables S1-S4) and acknowledge support from A. C. Magalhães, M. Almeida, D. Tuerker, S. Kuehl and C. Davies. This work was supported by the Biotechnology and Biological Sciences Research Council (BB/K006231/1 to M.S.), a Wellcome Trust Institutional Strategic Support Award (WT097835MF, WT105618MA to M.S.), the Portuguese Foundation for Science and Technology and FEDER/COMPETE (PTDC/BIA-BCM/118605/2010 to M.S.; SFRH/BD/37647/2007 to N.B.; SFRH/BPD/77619/2011 and UID/BIM/04501/2013 to D.R.). M.W., E.A.G., and M.S. are supported by Marie Curie Initial Training Network (ITN) action PerFuMe (316723)

The making of a mammalian peroxisome, version 2.0: mitochondria get into the mix

This is the author accepted manuscript. The final version is available from Nature Publishing Group via the DOI in this record.A recent report from the laboratory of Heidi McBride (McGill University) presents a role for mitochondria in the de novo biogenesis of peroxisomes in mammalian cells (1). Peroxisomes are essential organelles responsible for a wide variety of biochemical functions, from the generation of bile, to plasmalogen synthesis, reduction of peroxides, and the oxidation of very long chain fatty acids (2). Like mitochondria, peroxisomes proliferate primarily through growth and division of pre-existing peroxisomes (3-6). However, unlike mitochondria, peroxisomes do not fuse (5,7); further, and perhaps most importantly, they can also be born de novo, a process thought to occur through the generation of pre-peroxisomal vesicles that originate from the endoplasmic reticulum (reviewed in (8,9). De novo peroxisome biogenesis has been extensively studies in yeast, with a major focus on the role of the ER in this process. Comprehensive studies in mammalian cells are, however, scarce (5,10-12). By exploiting patient cells lacking mature peroxisomes, Sugiura et al. (1) now assign a role to ER and mitochondria in de novo mammalian peroxisome biogenesis by showing that the formation of immature preperoxisomes occurs through the fusion of Pex3- / Pex14-containing mitochondriaderived vesicles with Pex16-containing ER-derived vesicles

Combined T2 and diffusion-weighted MR Imaging with template prostate biopsies in men suspected with prostate cancer but negative transrectal ultrasound-guided biopsies

PURPOSE: Transperineal template prostate (TPB) biopsy has been shown to improve prostate cancer detection in men with rising PSA and previous negative TRUS biopsies. Diagnostic performance of this approach especially MR imaging and using reliable reference standard remains scantly reported. MATERIALS AND METHODS: A total of 200 patients, who were previously TRUS biopsy negative, were recruited in this study. All the participants had at least 28-core TPB under general anesthetic within 8 weeks of previous negative TRUS biopsies. In 15 men undergoing laparoscopic radical prostatectomy, prostate specimens were sectioned using custom-made molds and analyzed by experienced pathologist as a feasibility study. RESULTS: In total, 120 of 200 patients (60 %) had positive TPB biopsy results. All of these men had at least one negative biopsy from transrectal route. T2 diffusion-weighted MR imaging showed no lesion in almost one-third of these men (61/200; 30.5 %). Out of these, 33 (33/61; 54 %) showed malignancy on TPB including high-grade tumors (>Gleason 7). Out of 15 patients underwent surgery with a total of 52 lesions (mean 3.5) on radical prostatectomy histology analyses, TPB detected 36 (70 %) lesions only. Some of these lesions were Gleason 7 and more mostly located in the posterior basal area of prostate. CONCLUSIONS: Transperineal template biopsy technique is associated with significantly high prostate cancer detection rate in men with previous negative TRUS biopsies, however compared to radical prostatectomy histology map, a significant number of lesions can still be missed in the posterior and basal area of prostate

Giant peroxisomes in a moss (Physcomitrella patens) peroxisomal biogenesis factor 11 mutant

Peroxisomal biogenesis factor 11 (PEX11) proteins are found in yeasts, mammals and plants, and play a role in peroxisome morphology and regulation of peroxisome division. The moss Physcomitrella patens has six PEX11 isoforms which fall into two subfamilies, similar to those found in monocots and dicots. We carried out targeted gene disruption of the Phypa_PEX11-1 gene and compared the morphological and cellular phenotypes of the wild-type and mutant strains. The mutant grew more slowly and the development of gametophores was retarded. Mutant chloronemal filaments contained large cellular structures which excluded all other cellular organelles. Expression of fluorescent reporter proteins revealed that the mutant strain had greatly enlarged peroxisomes up to 10 μm in diameter. Expression of a vacuolar membrane marker confirmed that the enlarged structures were not vacuoles, or peroxisomes sequestered within vacuoles as a result of pexophagy. Phypa_PEX11 targeted to peroxisome membranes could rescue the knock out phenotype and interacted with Fission1 on the peroxisome membrane. Moss PEX11 functions in peroxisome division similar to PEX11 in other organisms but the mutant phenotype is more extreme and environmentally determined, making P. patens a powerful system in which to address mechanisms of peroxisome proliferation and division

POLRMT regulates the switch between replication primer formation and gene expression of mammalian mtDNA

Mitochondria are vital in providing cellular energy via their oxidative phosphorylation system, which requires the coordinated expression of genes encoded by both the nuclear and mitochondrial genomes (mtDNA). Transcription of the circular mammalian mtDNA depends on a single mitochondrial RNA polymerase (POLRMT). Although the transcription initiation process is well understood, it is debated whether POLRMT also serves as the primase for the initiation of mtDNA replication. In the nucleus, the RNA polymerases needed for gene expression have no such role. Conditional knockout of Polrmt in the heart results in severe mitochondrial dysfunction causing dilated cardiomyopathy in young mice. We further studied the molecular consequences of different expression levels of POLRMT and found that POLRMT is essential for primer synthesis to initiate mtDNA replication in vivo. Furthermore, transcription initiation for primer formation has priority over gene expression. Surprisingly, mitochondrial transcription factor A (TFAM) exists in an mtDNA-free pool in the Polrmt knockout mice. TFAM levels remain unchanged despite strong mtDNA depletion, and TFAM is thus protected from degradation of the AAA(+) Lon protease in the absence of POLRMT. Last, we report that mitochondrial transcription elongation factor may compensate for a partial depletion of POLRMT in heterozygous Polrmt knockout mice, indicating a direct regulatory role of this factor in transcription. In conclusion, we present in vivo evidence that POLRMT has a key regulatory role in the replication of mammalian mtDNA and is part of a transcriptional mechanism that provides a switch between primer formation for mtDNA replication and mitochondrial gene expression

Two randomized crossover multicenter studies investigating gastrointestinal symptoms after bread consumption in individuals with noncoeliac wheat sensitivity: do wheat species and fermentation type matter?

Background: Many individuals reduce their bread intake due to the belief that wheat is the cause of their gastrointestinal (GI) symptoms. Different grains and processing methods may impact tolerability. Objective: We investigated the effects of six different types of bread on GI symptoms in individuals with self-reported non-coeliac wheat sensitivity (NCWS). Methods: Two parallel randomised double-blind crossover multicentre studies were conducted. NCWS individuals, in whom coeliac disease and wheat allergy were ruled out, received five slices of (study A, n=20) yeast fermented (YF) or (study B, n=20) sourdough fermented (SF) bread made of bread wheat, spelt or emmer on three separate intervention days. Each test day was preceded by a run-in period of 3 days and separated by a wash-out period of at least 7 days. GI symptoms were evaluated by change in symptom score (test day minus average of the 3-day run-in period) on a 0-100mm visual analogue scale (delta VAS). Responders were defined as an increase in delta VAS of at least 15mm for overall GI symptoms, abdominal discomfort, abdominal pain, bloating and/or flatulence. Results: The overall change in GI symptoms did not differ between breads of different grains (YF p=0.267; SF p=0.144). The number of responders was also comparable for both YF (6 to wheat, 5 to spelt, and 7 to emmer, p=0.761) and SF breads (9 to wheat, 7 to spelt, and 8 to emmer, p=0.761). Conclusion: The majority of NCWS individuals experienced GI symptoms for at least one of the breads, but on a group level, no differences were found between different grain types for either YF or SF breads. Clinical Trial Registry: ClinicalTrials.gov, NCT0408447

Coastal lagoons and rising sea level: a review

Sea-level rise (SLR) poses a particularly ominous threat to human habitations and infrastructure in the coastal zone because 10% of the world's population lives in low-lying coastal regions within 10 m elevation of present sea level. There has been much discussion about projected (and the sources of projection) vs. measured SLR rates. Which rates should coastal scientists and managers apply in their studies, and what is the degree of confi- dence of such forecasts, are still open questions. This paper reviews the patterns and effects of relative SLR (RSLR) in coastal lagoons. Three main components are presented in the review: (a) a summary of the main approaches used in predicting medium- to long-term trends in RSLR, (b) a summary of the main evolutionary trends of coastal lagoons and the tools used to examine such trends, and (c) an identification of future research needs. The review reveals that the major source of uncertainty is how and when RSLR will manifest itself at different spatio-temporal scales in coastal lagoon systems, and how its effects can be mitigated. Most of the studies reviewed herein articulate a natural ‘defence’ mechanism of barriers in coastal lagoons by landward barrier retreat through continuous migration, and a gradual change in basin hypsometry during the retreat process. So far, only a relatively small number of detailed studies have integrated and quantified human impacts and coastal lagoon evolution induced by RSLR. We conclude that much more research about adaptation measures is needed, taking into consideration not only the physical and ecological systems but also social, cultural, and economic impacts. Future challenges include a downscaling of SLR approaches from the global level to regional and local levels, with a detailed application of coastal evolution prediction to individual coastal lagoon systemsinfo:eu-repo/semantics/publishedVersio