Biomedical subjects

Rohan D Teasdale

Publications and source records attributed to Rohan D Teasdale.

At least 19 recordsLinked to original sources

PubMed2006

Visualisation of macropinosome maturation by the recruitment of sorting nexins.

We report that phosphoinositol-binding sorting nexin 5 (SNX5) associates with newly formed macropinosomes induced by EGF stimulation. We used the recruitment of GFP-SNX5 to macropinosomes to track their maturation. Initially, GFP-SNX5 is sequestered to discrete subdomains of the macropinosome; these subdomains are subsequently incorporated into highly dynamic, often branched, tubular structures. Time-lapse videomicroscopy revealed the highly dynamic extension of SNX5-labelled tubules and their departure from the macropinosome body to follow predefined paths towards the perinuclear region of the cell, before fusing with early endosomal acceptor membranes. The extension and departure of these tubular structures occurs rapidly over 5-10 minutes and is dependent upon intact microtubules. As the tubular structures depart from the macropinosome there is a reduction in the surface area and an increase in tension of the limiting membrane of the macropinosome. In addition to the recruitment of SNX5 to the macropinosome, Rab5, SNX1 and EEA1 are also recruited by newly formed macropinosomes, followed by the accumulation of Rab7. SNX5 forms heterodimers with SNX1 and this interaction is required for endosome association of SNX5. We propose that the departure of SNX5-positive tubules represents a rapid mechanism of recycling components from macropinosomes thereby promoting their maturation into Rab7-positive structures. Collectively these findings provide a detailed real-time characterisation of the maturation process of the macropinocytic endosome.

Antigens, Surface↗

PubMed2006

Identification and analysis of novel genes expressed in the mouse embryonic facial primordia.

Craniofacial anomalies are a common feature of human congenital dysmorphology syndromes, suggesting that genes expressed in the developing face are likely to play a wider role in embryonic development. To facilitate the identification of genes involved in embryogenesis, we previously constructed an enriched cDNA library by subtracting adult mouse liver cDNA from that of embryonic day (E)10.5 mouse pharyngeal arch cDNA. From this library, 273 unique clones were sequenced and known proteins binned into functional categories in order to assess enrichment of the library (1). We have now selected 31 novel and poorly characterised genes from this library and present bioinformatic analysis to predict proteins encoded by these genes, and to detect evolutionary conservation. Of these genes 61% (19/31) showed restricted expression in the developing embryo, and a subset of these was chosen for further in silico characterisation as well as experimental determination of subcellular localisation based on transient transfection of predicted full-length coding sequences into mammalian cell lines. Where a human orthologue of these genes was detected, chromosomal localisation was determined relative to known loci for human congenital disease.

Animals↗

PubMed2006

Differential use of signal peptides and membrane domains is a common occurrence in the protein output of transcriptional units.

Membrane organization describes the orientation of a protein with respect to the membrane and can be determined by the presence, or absence, and organization within the protein sequence of two features: endoplasmic reticulum signal peptides and alpha-helical transmembrane domains. These features allow protein sequences to be classified into one of five membrane organization categories: soluble intracellular proteins, soluble secreted proteins, type I membrane proteins, type II membrane proteins, and multi-spanning membrane proteins. Generation of protein isoforms with variable membrane organizations can change a protein's subcellular localization or association with the membrane. Application of MemO, a membrane organization annotation pipeline, to the FANTOM3 Isoform Protein Sequence mouse protein set revealed that within the 8,032 transcriptional units (TUs) with multiple protein isoforms, 573 had variation in their use of signal peptides, 1,527 had variation in their use of transmembrane domains, and 615 generated protein isoforms from distinct membrane organization classes. The mechanisms underlying these transcript variations were analyzed. While TUs were identified encoding all pairwise combinations of membrane organization categories, the most common was conversion of membrane proteins to soluble proteins. Observed within our high-confidence set were 156 TUs predicted to generate both extracellular soluble and membrane proteins, and 217 TUs generating both intracellular soluble and membrane proteins. The differential use of endoplasmic reticulum signal peptides and transmembrane domains is a common occurrence within the variable protein output of TUs. The generation of protein isoforms that are targeted to multiple subcellular locations represents a major functional consequence of transcript variation within the mouse transcriptome.

Animals↗

PubMed2006

Spatial gene expression in the T-stage mouse metanephros.

The E11.5 mouse metanephros is comprised of a T-stage ureteric epithelial tubule sub-divided into tip and trunk cells surrounded by metanephric mesenchyme (MM). Tip cells are induced to undergo branching morphogenesis by the MM. In contrast, signals within the mesenchyme surrounding the trunk prevent ectopic branching of this region. In order to identify novel genes involved in the molecular regulation of branching morphogenesis we compared the gene expression profiles of isolated tip, trunk and MM cells using Compugen mouse long oligo microarrays. We identified genes enriched in the tip epithelium, sim-1, Arg2, Tacstd1, Crlf-1 and BMP7; genes enriched in the trunk epithelium, Innp1, Itm2b, Mkrn1, SPARC, Emu2 and Gsta3 and genes spatially restricted to the mesenchyme surrounding the trunk, CSPG2 and CV-2, with overlapping and complimentary expression to BMP4, respectively. This study has identified genes spatially expressed in regions of the developing kidney involved in branching morphogenesis, nephrogenesis and the development of the collecting duct system, calyces, renal pelvis and ureter.

Animals↗

PubMed2006

PhosphoregDB: the tissue and sub-cellular distribution of mammalian protein kinases and phosphatases.

BACKGROUND: Protein kinases and protein phosphatases are the fundamental components of phosphorylation dependent protein regulatory systems. We have created a database for the protein kinase-like and phosphatase-like loci of mouse http://phosphoreg.imb.uq.edu.au that integrates protein sequence, interaction, classification and pathway information with the results of a systematic screen of their sub-cellular localization and tissue specific expression data mined from the GNF tissue atlas of mouse. RESULTS: The database lets users query where a specific kinase or phosphatase is expressed at both the tissue and sub-cellular levels. Similarly the interface allows the user to query by tissue, pathway or sub-cellular localization, to reveal which components are co-expressed or co-localized. A review of their expression reveals 30% of these components are detected in all tissues tested while 70% show some level of tissue restriction. Hierarchical clustering of the expression data reveals that expression of these genes can be used to separate the samples into tissues of related lineage, including 3 larger clusters of nervous tissue, developing embryo and cells of the immune system. By overlaying the expression, sub-cellular localization and classification data we examine correlations between class, specificity and tissue restriction and show that tyrosine kinases are more generally expressed in fewer tissues than serine/threonine kinases. CONCLUSION: Together these data demonstrate that cell type specific systems exist to regulate protein phosphorylation and that for accurate modelling and for determination of enzyme substrate relationships the co-location of components needs to be considered.

Amino Acid Sequence↗

PubMed2006

Differential gene expression in the developing mouse ureter.

In many instances, kidney dysgenesis results as a secondary consequence to defects in the development of the ureter. Through the use of mouse genetics a number of genes associated with such malformations have been identified, however, the cause of many other abnormalities remain unknown. In order to identify novel genes involved in ureter development we compared gene expression in embryonic day (E) 12.5, E15.5 and postnatal day (P) 75 ureters using the Compugen mouse long oligo microarrays. A total of 248 genes were dynamically upregulated and 208 downregulated between E12.5 and P75. At E12.5, when the mouse ureter is comprised of a simple cuboidal epithelium surrounded by ureteric mesenchyme, genes previously reported to be expressed in the ureteric mesenchyme, foxC1 and foxC2 were upregulated. By E15.5 the epithelial layer develops into urothelium, impermeable to urine, and smooth muscle develops for the peristaltic movement of urine towards the bladder. The development of these two cell types coincided with the upregulation of UPIIIa, RAB27b and PPARgamma reported to be expressed in the urothelium, and several muscle genes, Acta1, Tnnt2, Myocd, and Tpm2. In situ hybridization identified several novel genes with spatial expression within the smooth muscle, Acta1; ureteric mesenchyme and smooth muscle, Thbs2 and Col5a2; and urothelium, Kcnj8 and Adh1. This study marks the first known report defining global gene expression of the developing mouse ureter and will provide insight into the molecular mechanisms underlying kidney and lower urinary tract malformations.

Animals↗

PubMed2006

LOCATE: a mouse protein subcellular localization database.

We present here LOCATE, a curated, web-accessible database that houses data describing the membrane organization and subcellular localization of proteins from the FANTOM3 Isoform Protein Sequence set. Membrane organization is predicted by the high-throughput, computational pipeline MemO. The subcellular locations of selected proteins from this set were determined by a high-throughput, immunofluorescence-based assay and by manually reviewing >1700 peer-reviewed publications. LOCATE represents the first effort to catalogue the experimentally verified subcellular location and membrane organization of mammalian proteins using a high-throughput approach and provides localization data for approximately 40% of the mouse proteome. It is available at http://locate.imb.uq.edu.au.

Animals↗

PubMed2006

Definition and spatial annotation of the dynamic secretome during early kidney development.

The term "secretome" has been defined as a set of secreted proteins (Grimmond et al. [2003] Genome Res 13:1350-1359). The term "secreted protein" encompasses all proteins exported from the cell including growth factors, extracellular proteinases, morphogens, and extracellular matrix molecules. Defining the genes encoding secreted proteins that change in expression during organogenesis, the dynamic secretome, is likely to point to key drivers of morphogenesis. Such secreted proteins are involved in the reciprocal interactions between the ureteric bud (UB) and the metanephric mesenchyme (MM) that occur during organogenesis of the metanephros. Some key metanephric secreted proteins have been identified, but many remain to be determined. In this study, microarray expression profiling of E10.5, E11.5, and E13.5 kidney and consensus bioinformatic analysis were used to define a dynamic secretome of early metanephric development. In situ hybridisation was used to confirm microarray results and clarify spatial expression patterns for these genes. Forty-one secreted factors were dynamically expressed between the E10.5 and E13.5 timeframe profiled, and 25 of these factors had not previously been implicated in kidney development. A text-based anatomical ontology was used to spatially annotate the expression pattern of these genes in cultured metanephric explants.

Animals↗

PubMed2006

Predicting the solvent accessibility of transmembrane residues from protein sequence.

In this study, we propose a novel method to predict the solvent accessible surface areas of transmembrane residues. For both transmembrane alpha-helix and beta-barrel residues, the correlation coefficients between the predicted and observed accessible surface areas are around 0.65. On the basis of predicted accessible surface areas, residues exposed to the lipid environment or buried inside a protein can be identified by using certain cutoff thresholds. We have extensively examined our approach based on different definitions of accessible surface areas and a variety of sets of control parameters. Given that experimentally determining the structures of membrane proteins is very difficult and membrane proteins are actually abundant in nature, our approach is useful for theoretically modeling membrane protein tertiary structures, particularly for modeling the assembly of transmembrane domains. This approach can be used to annotate the membrane proteins in proteomes to provide extra structural and functional information.

Cell Membrane↗

PubMed2006

Subcellular localization of mammalian type II membrane proteins.

Application of a computational membrane organization prediction pipeline, MemO, identified putative type II membrane proteins as proteins predicted to encode a single alpha-helical transmembrane domain (TMD) and no signal peptides. MemO was applied to RIKEN's mouse isoform protein set to identify 1436 non-overlapping genomic regions or transcriptional units (TUs), which encode exclusively type II membrane proteins. Proteins with overlapping predicted InterPro and TMDs were reviewed to discard false positive predictions resulting in a dataset comprised of 1831 transcripts in 1408 TUs. This dataset was used to develop a systematic protocol to document subcellular localization of type II membrane proteins. This approach combines mining of published literature to identify subcellular localization data and a high-throughput, polymerase chain reaction (PCR)-based approach to experimentally characterize subcellular localization. These approaches have provided localization data for 244 and 169 proteins. Type II membrane proteins are localized to all major organelle compartments; however, some biases were observed towards the early secretory pathway and punctate structures. Collectively, this study reports the subcellular localization of 26% of the defined dataset. All reported localization data are presented in the LOCATE database (http://www.locate.imb.uq.edu.au).

Animals↗

PubMed2006

Transcript annotation in FANTOM3: mouse gene catalog based on physical cDNAs.

The international FANTOM consortium aims to produce a comprehensive picture of the mammalian transcriptome, based upon an extensive cDNA collection and functional annotation of full-length enriched cDNAs. The previous dataset, FANTOM2, comprised 60,770 full-length enriched cDNAs. Functional annotation revealed that this cDNA dataset contained only about half of the estimated number of mouse protein-coding genes, indicating that a number of cDNAs still remained to be collected and identified. To pursue the complete gene catalog that covers all predicted mouse genes, cloning and sequencing of full-length enriched cDNAs has been continued since FANTOM2. In FANTOM3, 42,031 newly isolated cDNAs were subjected to functional annotation, and the annotation of 4,347 FANTOM2 cDNAs was updated. To accomplish accurate functional annotation, we improved our automated annotation pipeline by introducing new coding sequence prediction programs and developed a Web-based annotation interface for simplifying the annotation procedures to reduce manual annotation errors. Automated coding sequence and function prediction was followed with manual curation and review by expert curators. A total of 102,801 full-length enriched mouse cDNAs were annotated. Out of 102,801 transcripts, 56,722 were functionally annotated as protein coding (including partial or truncated transcripts), providing to our knowledge the greatest current coverage of the mouse proteome by full-length cDNAs. The total number of distinct non-protein-coding transcripts increased to 34,030. The FANTOM3 annotation system, consisting of automated computational prediction, manual curation, and final expert curation, facilitated the comprehensive characterization of the mouse transcriptome, and could be applied to the transcriptomes of other species.

Animals↗

PubMed2005

Prediction of protein B-factor profiles.

The polypeptide backbones and side chains of proteins are constantly moving due to thermal motion and the kinetic energy of the atoms. The B-factors of protein crystal structures reflect the fluctuation of atoms about their average positions and provide important information about protein dynamics. Computational approaches to predict thermal motion are useful for analyzing the dynamic properties of proteins with unknown structures. In this article, we utilize a novel support vector regression (SVR) approach to predict the B-factor distribution (B-factor profile) of a protein from its sequence. We explore schemes for encoding sequences and various settings for the parameters used in SVR. Based on a large dataset of high-resolution proteins, our method predicts the B-factor distribution with a Pearson correlation coefficient (CC) of 0.53. In addition, our method predicts the B-factor profile with a CC of at least 0.56 for more than half of the proteins. Our method also performs well for classifying residues (rigid vs. flexible). For almost all predicted B-factor thresholds, prediction accuracies (percent of correctly predicted residues) are greater than 70%. These results exceed the best results of other sequence-based prediction methods.

Algorithms↗

PubMed2005

Polarized trafficking of E-cadherin is regulated by Rac1 and Cdc42 in Madin-Darby canine kidney cells.

E-cadherin is a major cell-cell adhesion protein of epithelia that is trafficked to the basolateral cell surface in a polarized fashion. The exact post-Golgi route and regulation of E-cadherin transport have not been fully described. The Rho GTPases Cdc42 and Rac1 have been implicated in many cell functions, including the exocytic trafficking of other proteins in polarized epithelial cells. These Rho family proteins are also associated with the cadherin-catenin complexes at the cell surface. We have used functional mutants of Rac1 and Cdc42 and inactivating toxins to demonstrate specific roles for both Cdc42 and Rac1 in the post-Golgi transport of E-cadherin. Dominant-negative mutants of Cdc42 and Rac1 accumulate E-cadherin at a distinct post-Golgi step. This accumulation occurs before p120(ctn) interacts with E-cadherin, because p120(ctn) localization was not affected by the Cdc42 or Rac1 mutants. Moreover, the GTPase mutants had no effect on the trafficking of a targeting mutant of E-cadherin, consistent with the selective involvement of Cdc42 and Rac1 in basolateral trafficking. These results provide a new example of Rho GTPase regulation of basolateral trafficking and demonstrate novel roles for Cdc42 and Rac1 in the post-Golgi transport of E-cadherin.

Animals↗

PubMed2005

A novel hook-related protein family and the characterization of hook-related protein 1.

The spatial organization of organelles within a cell is dependent on microtubules. Recently, members of the Hook family of proteins have been proposed to function in linking organelles to microtubules. We report the identification of a completely novel protein family, the Hook-related protein (HkRP) family, from which the Hook proteins have diverged. Bioinformatic analysis of the HkRP family revealed several conserved domains, including a unique C-terminal HkRP domain. The central region of each protein is comprised of an extensive coiled-coil domain, and the N-terminus contains a putative microtubule-binding domain. This domain has been shown to bind microtubules in the Hook protein and show that the HkRP1 protein is microtubule-associated. While endogenous HkRP1 has no distinct organelle association, expression of the C-terminal membrane-binding domain suggests a function of the HkRP1 in early endosome. Ultrastructural studies reveal that expression of the C-terminal HkRP1 domain causes an accumulation of internal membranes with an electron-dense coat. Co-localization studies show a concomitant redistribution of the early endosome marker sorting-nexin 1 but not the early endosome antigen-1 (EEA1). The steady-state distribution of the epidermal growth factor receptor is also specifically disrupted by expression of the C-terminal domain. We propose that HkRP1 is involved in the process of tubulation of sorting nexin-1 positive membranes from early endosome subdomains.

Amino Acid Sequence↗

PubMed2005

A novel mammalian retromer component, Vps26B.

The mammalian retromer protein complex, which consists of three proteins--Vps26, Vps29, and Vps35--in association with members of the sorting nexin family of proteins, has been implicated in the trafficking of receptors and their ligands within the endosomal/lysosomal system of mammalian cells. A bioinformatic analysis of the mouse genome identified an additional transcribed paralog of the Vps26 retromer protein, which we termed Vps26B. No paralogs were identified for Vps29 and Vps35. Phylogenetic studies indicate that the two paralogs of Vps26 become evident after the evolution of the chordates. We propose that the chordate Vps26-like gene published previously be renamed Vps26A to differentiate it from Vps26B. As for Vps26A, biochemical characterization of Vps26B established that this novel 336 amino acid residue protein is a peripheral membrane protein. Vps26B co-precipitated with Vps35 from transfected cells and the direct interaction between these two proteins was confirmed by yeast 2-hybrid analysis, thereby establishing Vps26B as a subunit of the retromer complex. Within HeLa cells, Vps26B was found in the cytoplasm with low levels at the plasma membrane, while Vps26A was predominantly associated with endosomal membranes. Within A549 cells, both Vps26A and Vps26B co-localized with actin-rich lamellipodia at the cell surface. These structures also co-localized with Vps35. Total internal reflection fluorescence microscopy confirmed the association of Vps26B with the plasma membrane in a stable HEK293 cell line expressing cyan fluorescent protein (CFP)-Vps26B. Based on these observations, we propose that the mammalian retromer complex is located at both endosomes and the plasma membrane in some cell types.

Amino Acid Sequence↗

PubMed2005

In vivo analysis of growth hormone receptor signaling domains and their associated transcripts.

The growth hormone receptor (GHR) is a critical regulator of postnatal growth and metabolism. However, the GHR signaling domains and pathways that regulate these processes in vivo are not defined. We report the first knock-in mouse models with deletions of specific domains of the receptor that are required for its in vivo actions. Mice expressing truncations at residue m569 (plus Y539/545-F) and at residue m391 displayed a progressive impairment of postnatal growth with receptor truncation. Moreover, after 4 months of age, marked male obesity was observed in both mutant 569 and mutant 391 and was associated with hyperglycemia. Both mutants activated hepatic JAK2 and ERK2, whereas STAT5 phosphorylation was substantially decreased for mutant 569 and absent from mutant 391, correlating with loss of IGF-1 expression and reduction in growth. Microarray analysis of these and GHR(-/-) mice demonstrated that particular signaling domains are responsible for the regulation of different target genes and revealed novel actions of growth hormone. These mice represent the first step in delineating the domains of the GHR regulating body growth and composition and the transcripts associated with these domains.

Animals↗

PubMed2004

Sorting nexin 5 is localized to a subdomain of the early endosomes and is recruited to the plasma membrane following EGF stimulation.

Sorting nexins are a large family of proteins that contain the phosphoinositide-binding Phox homology (PX) domain. A number of sorting nexins are known to bind to PtdIns3P, which mediates their localization to membranes of the endocytic pathway. We show here that sorting nexin 5 (SNX5) can be recruited to two distinct membrane compartments. In non-stimulated cells, the PX domain was independently targeted to endosomal structures and colocalized with full-length SNX5. The membrane binding of the PX domain was inhibited by the PI 3-kinase inhibitor, wortmannin. Although SNX5 colocalized with a fluid-phase marker and was found predominantly within a PtdIns3P-rich endosomal domain, very little colocalization was observed between SNX5 and the PtdIns3P-binding protein, EEA1. Using liposome-based binding assays, we have shown that the PX domain of SNX5 interacts not only with PtdIns3P but also with PtdIns3,4P2. In response to EGF stimulation, either the SNX5-PX domain or full-length SNX5 was rapidly recruited to the plasma membrane. The localization of SNX1, which does not bind PtdIns3,4P2, was unaffected by EGF signalling. Therefore, SNX5 is localized to a subdomain of the early endosome distinct from EEA1 and, following EGF stimulation and elevation of PtdIns3,4P2, is also transiently recruited to the plasma membrane. These results indicate that SNX5 may have functions not only associated with endosomal sorting but also with the phosphoinositide-signalling pathway.

Androstadienes↗

PubMed2004

Identification of a Golgi-localised GRIP domain protein from Arabidopsis thaliana.

A family of Golgi-localised molecules was recently described in animals and fungi possessing extensive coiled regions and a short (approximately 40 residues) conserved C-terminal domain, called the GRIP domain, which is responsible for their location to this organelle. Using the model plant Arabidopsis thaliana, we identified a gene (AtGRIP) encoding a putative GRIP protein. We demonstrated that the C-terminal domain from AtGRIP functions as a Golgi-targeting sequence in plant cells. Localisation studies in living cells expressing the AtGRIP fused to a DsRed2 fluorescent probe, showed extensive co-location with the Golgi marker alpha-mannosidase I in transformed tobacco protoplasts. GRIP-like sequences were also found in genomic databases of rice, maize, wheat and alfalfa, suggesting that this domain may be a useful Golgi marker for immunolocalisation studies. Despite low sequence identity amongst GRIP domains, the plant GRIP sequence was able to target to the Golgi of mammalian cells. Taken together, these data indicate that GRIP domain proteins might be implicated in a targeting mechanism that is conserved amongst eukaryotes.

Amino Acid Sequence↗