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FOP Is a Centriolar Satellite Protein Involved in Ciliogenesis  [PDF]
Joanna Y. Lee, Tim Stearns
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0058589
Abstract: Centriolar satellites are proteinaceous granules that are often clustered around the centrosome. Although centriolar satellites have been implicated in protein trafficking in relation to the centrosome and cilium, the details of their function and composition remain unknown. FOP (FGFR1 Oncogene Partner) is a known centrosome protein with homology to the centriolar satellite proteins FOR20 and OFD1. We find that FOP partially co-localizes with the satellite component PCM1 in a cell cycle-dependent manner, similarly to the satellite and cilium component BBS4. As for BBS4, FOP localization to satellites is cell cycle dependent, with few satellites labeled in G1, when FOP protein levels are lowest, and most labeled in G2. FOP-FGFR1, an oncogenic fusion that causes a form of leukemia called myeloproliferative neoplasm, also localizes to centriolar satellites where it increases tyrosine phosphorylation. Depletion of FOP strongly inhibits primary cilium formation in human RPE-1 cells. These results suggest that FOP is a centriolar satellite cargo protein and, as for several other satellite-associated proteins, is involved in ciliogenesis. Localization of the FOP-FGFR1 fusion kinase to centriolar satellites may be relevant to myeloproliferative neoplasm disease progression.
Expression and Purification of Chaperone-Active Recombinant Clusterin  [PDF]
Rebecca A. Dabbs, Mark R. Wilson
PLOS ONE , 2014, DOI: 10.1371/journal.pone.0086989
Abstract: Clusterin was the first described secreted mammalian chaperone and is implicated as being a key player in both intra- and extracellular proteostasis. Its unique combination of structural features and biological chaperone activity has, however, previously made it very challenging to express and purify the protein in a correctly processed and chaperone-active form. While there are multiple reports in the literature describing the use of recombinant clusterin, all of these reports suffer from one or more of the following shortcomings: details of the methods used to produce the protein are poorly described, the product is incompletely (if at all) characterised, and purity (if shown) is in many cases inadequate. The current report provides the first well validated method to economically produce pure chaperone-active recombinant clusterin. The method was developed after trialling expression in cultured bacterial, yeast, insect and mammalian cells, and involves the expression of recombinant clusterin from stably transfected HEK293 cells in protein-free medium. The product is expressed at between 7.5 and 10 μg/ml of culture, and is readily purified by a combination of immunoaffinity, cation exchange and size exclusion chromatography. The purified product was shown to be glycosylated, correctly proteolytically cleaved into α- and β-subunits, and have chaperone activity similar to that of human plasma clusterin. This new method creates the opportunity to use mutagenesis and metabolic labelling approaches in future studies to delineate functionally important sites within clusterin, and also provides a theoretically unlimited supply of recombinant clusterin which may in the future find applications in the development of therapeutics.
The architecture of mammalian ribosomal protein promoters
Robert P Perry
BMC Evolutionary Biology , 2005, DOI: 10.1186/1471-2148-5-15
Abstract: A striking evolutionarily conserved feature of most rp genes is the separation by an intron of the sequences involved in transcriptional and translational regulation from the sequences with protein encoding function. Another conserved feature is the polypyrimidine initiator, which conforms to the consensus (Y)2C+1TY(T)2(Y)3. At least 60 % of the rp promoters contain a largely conserved TATA box or A/T-rich motif, which should theoretically have TBP-binding capability. A remarkably high proportion of the promoters contain conserved binding sites for transcription factors that were previously implicated in rp gene expression, namely upstream GABP and Sp1 sites and downstream YY1 sites. Over 80 % of human and mouse rp genes contain a transposable element residue within 900 bp of 5' flanking sequence; very little sequence identity between human and mouse orthologues was evident more than 200 bp upstream of the transcriptional start point.This analysis has provided some valuable insights into the general architecture of mammalian rp promoters and has identified parameters that might coordinately regulate the transcriptional activity of certain subsets of rp genes.Ribosomes are vital organelles, which catalyze protein synthesis in all living organisms. Eukaryotic ribosomes consist of four RNA molecules (rRNAs) and 79 different proteins. The mammalian genes encoding the rRNAs are multicopy and clustered at a few loci, whereas those encoding the ribosomal proteins (rp genes) are single copy and scattered throughout the genome [1]. In addition to the functional rp genes, all of which contain introns, mammalian genomes contain many nonfunctional intronless rp pseudogenes [2]. The earliest determinations of mouse rp gene sequences and of transcriptional start points (tsp's) revealed a salient feature of rp genes, namely that transcription is initiated at a C residue within a polypyrimidine tract [3-5]. A recent study by Kenmochi and coworkers [6] has demonstrated that this is
Localization of a Guanylyl Cyclase to Chemosensory Cilia Requires the Novel Ciliary MYND Domain Protein DAF-25  [PDF]
Victor L. Jensen,Nathan J. Bialas,Sharon L. Bishop-Hurley,Laurie L. Molday,Katarzyna Kida,Phuong Anh T. Nguyen,Oliver E. Blacque,Robert S. Molday,Michel R. Leroux,Donald L. Riddle
PLOS Genetics , 2010, DOI: 10.1371/journal.pgen.1001199
Abstract: In harsh conditions, Caenorhabditis elegans arrests development to enter a non-aging, resistant diapause state called the dauer larva. Olfactory sensation modulates the TGF-β and insulin signaling pathways to control this developmental decision. Four mutant alleles of daf-25 (abnormal DAuer Formation) were isolated from screens for mutants exhibiting constitutive dauer formation and found to be defective in olfaction. The daf-25 dauer phenotype is suppressed by daf-10/IFT122 mutations (which disrupt ciliogenesis), but not by daf-6/PTCHD3 mutations (which prevent environmental exposure of sensory cilia), implying that DAF-25 functions in the cilia themselves. daf-25 encodes the C. elegans ortholog of mammalian Ankmy2, a MYND domain protein of unknown function. Disruption of DAF-25, which localizes to sensory cilia, produces no apparent cilia structure anomalies, as determined by light and electron microscopy. Hinting at its potential function, the dauer phenotype, epistatic order, and expression profile of daf-25 are similar to daf-11, which encodes a cilium-localized guanylyl cyclase. Indeed, we demonstrate that DAF-25 is required for proper DAF-11 ciliary localization. Furthermore, the functional interaction is evolutionarily conserved, as mouse Ankmy2 interacts with guanylyl cyclase GC1 from ciliary photoreceptors. The interaction may be specific because daf-25 mutants have normally-localized OSM-9/TRPV4, TAX-4/CNGA1, CHE-2/IFT80, CHE-11/IFT140, CHE-13/IFT57, BBS-8, OSM-5/IFT88, and XBX-1/D2LIC in the cilia. Intraflagellar transport (IFT) (required to build cilia) is not defective in daf-25 mutants, although the ciliary localization of DAF-25 itself is influenced in che-11 mutants, which are defective in retrograde IFT. In summary, we have discovered a novel ciliary protein that plays an important role in cGMP signaling by localizing a guanylyl cyclase to the sensory organelle.
Evolutionarily Conserved Pattern of AMPA Receptor Subunit Glycosylation in Mammalian Frontal Cortex  [PDF]
Janusz Tucholski, Anita L. Pinner, Micah S. Simmons, James H. Meador-Woodruff
PLOS ONE , 2014, DOI: 10.1371/journal.pone.0094255
Abstract: Protein glycosylation may contribute to the evolution of mammalian brain complexity by adapting excitatory neurotransmission in response to environmental and social cues. Balanced excitatory synaptic transmission is primarily mediated by glutamatergic neurotransmission. Previous studies have found that subunits of the AMPA subtype of glutamate receptor are N-glycosylated, which may play a critical role in AMPA receptor trafficking and function at the cell membrane. Studies have predominantly used rodent models to address altered glycosylation in human pathological conditions. Given the rate of mammalian brain evolution and the predicted rate of change in the brain-specific glycoproteome, we asked if there are species-specific changes in glycoprotein expression, focusing on the AMPA receptor. N-glycosylation of AMPA receptor subunits was investigated in rat (Rattus norvegicus), tree shrew (Tupaia glis belangeri), macaque (Macaca nemestrina), and human frontal cortex tissue using a combination of enzymatic deglycosylation and Western blot analysis, as well as lectin binding assays. We found that two AMPA receptor subunits, GluA2 and GluA4, are sensitive to deglycosylation with Endo H and PNGase F. When we enriched for glycosylated proteins using lectin binding assays, we found that all four AMPA receptor subunits are glycosylated, and were predominantly recognized by lectins that bind to glucose or mannose, N-acetylglucosamine (GlcNAc), or 1-6αfucose. We found differences in glycosylation between different subunits, as well as modest differences in glycosylation of homologous subunits between different species.
Development of reverse phase protein microarrays for the validation of clusterin, a mid-abundant blood biomarker
Adriana Aguilar-Mahecha, Christiane Cantin, Maureen O'Connor-McCourt, Andre Nantel, Mark Basik
Proteome Science , 2009, DOI: 10.1186/1477-5956-7-15
Abstract: In the present study we present the development of reverse-phase protein microarrays (RPPMs) for the measurement of clusterin, a mid-abundant blood biomarker. An experimental protocol was optimized for the printing of serum and plasma on RPPMs using epoxy coated microscope slides and a non-denaturing printing buffer. Using fluorescent-tagged secondary antibodies, we achieved the reproducible detection of clusterin in spotted serum and plasma and reached a limit of detection of 780 ng/mL. Validation studies using both spiked clusterin and clinical samples showed excellent correlations with ELISA measurements of clusterin.Serum and plasma spotted in the reverse phase array format allow for reliable and reproducible high-throughput validation of a mid-abundant blood biomarker such as clusterin.The increasing application of genomics and proteomics technologies in medical research is making possible the development of "personalized medicine", i.e. medical care characterized by the use of biomarkers for the molecular diagnosis of different disease states and for the selection of therapies tailored to the individual's disease. Although both tissue and blood biomarkers are being discovered, blood derived-biomarkers are particularly attractive in the clinic since blood collection is inexpensive and relatively non-invasive and blood comes in contact with all tissues in the body. Unfortunately, the translation of putative blood biomarkers into clinical application has been hindered by the lack of a high-throughput technical platform for their validation. ELISAs are the standard method currently used for blood biomarker validation. However, the requirement for large sample volumes (~100 μl) and its low throughput make ELISA a costly method, both in time and biological material, not suited for the rapid validation of clinical samples.A novel technology designed to measure protein levels in a high-throughput fashion is the protein microarray [1,2]. There are two main types of pro
A Role for Alstr?m Syndrome Protein, Alms1, in Kidney Ciliogenesis and Cellular Quiescence  [PDF]
Guochun Li,Raquel Vega,Keats Nelms,Nicholas Gekakis,Christopher Goodnow,Peter McNamara,Hua Wu,Nancy A Hong,Richard Glynne
PLOS Genetics , 2007, DOI: 10.1371/journal.pgen.0030008
Abstract: Premature truncation alleles in the ALMS1 gene are a frequent cause of human Alstr?m syndrome. Alstr?m syndrome is a rare disorder characterized by early obesity and sensory impairment, symptoms shared with other genetic diseases affecting proteins of the primary cilium. ALMS1 localizes to centrosomes and ciliary basal bodies, but truncation mutations in Alms1/ALMS1 do not preclude formation of cilia. Here, we show that in vitro knockdown of Alms1 in mice causes stunted cilia on kidney epithelial cells and prevents these cells from increasing calcium influx in response to mechanical stimuli. The stunted-cilium phenotype can be rescued with a 5′ fragment of the Alms1 cDNA, which resembles disease-associated alleles. In a mouse model of Alstr?m syndrome, Alms1 protein can be stably expressed from the mutant allele and is required for cilia formation in primary cells. Aged mice developed specific loss of cilia from the kidney proximal tubules, which is associated with foci of apoptosis or proliferation. As renal failure is a common cause of mortality in Alstr?m syndrome patients, we conclude that this disease should be considered as a further example of the class of renal ciliopathies: wild-type or mutant alleles of the Alstr?m syndrome gene can support normal kidney ciliogenesis in vitro and in vivo, but mutant alleles are associated with age-dependent loss of kidney primary cilia.
Wdpcp, a PCP Protein Required for Ciliogenesis, Regulates Directional Cell Migration and Cell Polarity by Direct Modulation of the Actin Cytoskeleton  [PDF]
Cheng Cui,Bishwanath Chatterjee,Thomas P. Lozito,Zhen Zhang,Richard J. Francis,Hisato Yagi,Lisa M. Swanhart,Subramaniam Sanker,Deanne Francis,Qing Yu,Jovenal T. San Agustin,Chandrakala Puligilla,Tania Chatterjee,Terry Tansey,Xiaoqin Liu,Matthew W. Kelley,Elias T. Spiliotis,Adam V. Kwiatkowski,Rocky Tuan,Gregory J. Pazour,Neil A. Hukriede,Cecilia W. Lo
PLOS Biology , 2013, DOI: 10.1371/journal.pbio.1001720
Abstract: Planar cell polarity (PCP) regulates cell alignment required for collective cell movement during embryonic development. This requires PCP/PCP effector proteins, some of which also play essential roles in ciliogenesis, highlighting the long-standing question of the role of the cilium in PCP. Wdpcp, a PCP effector, was recently shown to regulate both ciliogenesis and collective cell movement, but the underlying mechanism is unknown. Here we show Wdpcp can regulate PCP by direct modulation of the actin cytoskeleton. These studies were made possible by recovery of a Wdpcp mutant mouse model. Wdpcp-deficient mice exhibit phenotypes reminiscent of Bardet–Biedl/Meckel–Gruber ciliopathy syndromes, including cardiac outflow tract and cochlea defects associated with PCP perturbation. We observed Wdpcp is localized to the transition zone, and in Wdpcp-deficient cells, Sept2, Nphp1, and Mks1 were lost from the transition zone, indicating Wdpcp is required for recruitment of proteins essential for ciliogenesis. Wdpcp is also found in the cytoplasm, where it is localized in the actin cytoskeleton and in focal adhesions. Wdpcp interacts with Sept2 and is colocalized with Sept2 in actin filaments, but in Wdpcp-deficient cells, Sept2 was lost from the actin cytoskeleton, suggesting Wdpcp is required for Sept2 recruitment to actin filaments. Significantly, organization of the actin filaments and focal contacts were markedly changed in Wdpcp-deficient cells. This was associated with decreased membrane ruffling, failure to establish cell polarity, and loss of directional cell migration. These results suggest the PCP defects in Wdpcp mutants are not caused by loss of cilia, but by direct disruption of the actin cytoskeleton. Consistent with this, Wdpcp mutant cochlea has normal kinocilia and yet exhibits PCP defects. Together, these findings provide the first evidence, to our knowledge, that a PCP component required for ciliogenesis can directly modulate the actin cytoskeleton to regulate cell polarity and directional cell migration.
Evolutionarily Conserved Herpesviral Protein Interaction Networks  [PDF]
Even Fossum,Caroline C. Friedel,Seesandra V. Rajagopala,Bj?rn Titz,Armin Baiker,Tina Schmidt,Theo Kraus,Thorsten Stellberger,Christiane Rutenberg,Silpa Suthram,Sourav Bandyopadhyay,Dietlind Rose,Albrecht von Brunn,Mareike Uhlmann,Christine Zeretzke,Yu-An Dong,Hélène Boulet,Manfred Koegl,Susanne M. Bailer,Ulrich Koszinowski,Trey Ideker,Peter Uetz,Ralf Zimmer,Jürgen Haas
PLOS Pathogens , 2009, DOI: 10.1371/journal.ppat.1000570
Abstract: Herpesviruses constitute a family of large DNA viruses widely spread in vertebrates and causing a variety of different diseases. They possess dsDNA genomes ranging from 120 to 240 kbp encoding between 70 to 170 open reading frames. We previously reported the protein interaction networks of two herpesviruses, varicella-zoster virus (VZV) and Kaposi's sarcoma-associated herpesvirus (KSHV). In this study, we systematically tested three additional herpesvirus species, herpes simplex virus 1 (HSV-1), murine cytomegalovirus and Epstein-Barr virus, for protein interactions in order to be able to perform a comparative analysis of all three herpesvirus subfamilies. We identified 735 interactions by genome-wide yeast-two-hybrid screens (Y2H), and, together with the interactomes of VZV and KSHV, included a total of 1,007 intraviral protein interactions in the analysis. Whereas a large number of interactions have not been reported previously, we were able to identify a core set of highly conserved protein interactions, like the interaction between HSV-1 UL33 with the nuclear egress proteins UL31/UL34. Interactions were conserved between orthologous proteins despite generally low sequence similarity, suggesting that function may be more conserved than sequence. By combining interactomes of different species we were able to systematically address the low coverage of the Y2H system and to extract biologically relevant interactions which were not evident from single species.
Are residues in a protein folding nucleus evolutionarily conserved?  [PDF]
Yan Yuan Tseng,Jie Liang
Quantitative Biology , 2003,
Abstract: It is important to understand how protein folding and evolution influences each other. Several studies based on entropy calculation correlating experimental measurement of residue participation in folding nucleus and sequence conservation have reached different conclusions. Here we report analysis of conservation of folding nucleus using an evolutionary model alternative to entropy based approaches. We employ a continuous time Markov model of codon substitution to distinguish mutation fixed by evolution and mutation fixed by chance. This model takes into account bias in codon frequency, bias favoring transition over transversion, as well as explicit phylogenetic information. We measure selection pressure using the ratio $\omega$ of synonymous vs. non-synonymous substitution at individual residue site. The $\omega$-values are estimated using the {\sc Paml} method, a maximum-likelihood estimator. Our results show that there is little correlation between the extent of kinetic participation in protein folding nucleus as measured by experimental $\phi$-value and selection pressure as measured by $\omega$-value. In addition, two randomization tests failed to show that folding nucleus residues are significantly more conserved than the whole protein. These results suggest that at the level of codon substitution, there is no indication that folding nucleus residues are significantly more conserved than other residues. We further reconstruct candidate ancestral residues of the folding nucleus and suggest possible test tube mutation studies of ancient folding nucleus.
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