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Biomedical subjects

Judith E Sleeman

Publications and source records attributed to Judith E Sleeman.

6 recordsLinked to original sources
PubMed2006

Molecular and functional characterization of microsomal UDP-glucuronic acid uptake by members of the nucleotide sugar transporter (NST) family.

Transport of the co-substrate UDPGA (UDP-glucuronic acid) into the lumen of the endoplasmic reticulum is an essential step in glucuronidation reactions due to the intraluminal location of the catalytic site of the enzyme UGT (UDP-glucuronosyltransferase). In the present study, we have characterized the function of several NSTs (nucleotide sugar transporters) and UGTs as potential carriers of UDPGA for glucuronidation reactions. UDPGlcNAc (UDP-N-acetylglucosamine)-dependent UDPGA uptake was found both in rat liver microsomes and in microsomes prepared from the rat hepatoma cell line H4IIE. The latency of UGT activity in microsomes derived from rat liver and V79 cells expressing UGT1A6 correlated well with mannose-6-phosphatase latency, confirming the UGT in the recombinant cells retained a physiology similar to rat liver microsomes. In the present study, four cDNAs coding for NSTs were obtained; two were previously reported (UGTrel1 and UGTrel7) and two newly identified (huYEA4 and huYEA4S). Localization of NSTs within the human genome sequence revealed that huYEA4S is an alternatively spliced form of huYEA4. All the cloned NSTs were stably expressed in V79 (Chinese hamster fibroblast) cells, and were able to transport UDPGA after preloading of isolated microsomal vesicles with UDPGlcNAc. The highest uptake was seen with UGTrel7, which displayed a V(max) approx. 1% of rat liver microsomes. Treatment of H4IIE cells with beta-naphthoflavone induced UGT protein expression but did not affect the rate of UDPGA uptake. Furthermore, microsomes from UGT1-deficient Gunn rat liver showed UDPGA uptake similar to those from control rats. These data show that NSTs can act as UDPGA transporters for glucuronidation reactions, and indicate that UGTs of the 1A family do not function as UDPGA carriers in microsomes. The cell line H4IIE is a useful model for the study of UDPGA transporters for glucuronidation reactions.

Animals↗
PubMed2005

FRET analyses of the U2AF complex localize the U2AF35/U2AF65 interaction in vivo and reveal a novel self-interaction of U2AF35.

We have analyzed the interaction between the U2AF subunits U2AF35 and U2AF65 in vivo using fluorescence resonance energy transfer (FRET) microscopy. U2 snRNP Auxiliary Factor (U2AF) is an essential pre-mRNA splicing factor complex, comprising 35-kDa (U2AF35) and 65-kDa (U2AF65) subunits. U2AF65 interacts directly with the polypyrimidine tract and promotes binding of U2 snRNP to the pre-mRNA branchpoint, while U2AF35 associates with the conserved AG dinucleotide at the 3' end of the intron and has multiple functions in the splicing process. Using two different approaches for measuring FRET, we have identified and spatially localized sites of direct interaction between U2AF35 and U2AF65 in vivo in live cell nuclei. While U2AF is thought to function as a heterodimeric complex, the FRET data have also revealed a novel U2AF35 self-interaction in vivo, which is confirmed in vitro using biochemical assays. These results suggest that the stoichiometry of the U2AF complex may, at least in part, differ in vivo from the expected heterodimeric complex. The data show that FRET studies offer a valuable approach for probing interactions between pre-mRNA splicing factors in vivo.

Bacterial Proteins↗
PubMed2004

Dynamics of the mammalian nucleus: can microscopic movements help us to understand our genes?

The cell is the basic building block of human life. Each of us has existed as a single cell--the fertilized egg--and each of us is made up of billions of cells specialized in many different ways to form our tissues and organs. The nucleus of the cell, described as far back as 1682, is known to be the site of storage of chromosomes that carry the essential and unique DNA blueprint for life. With the recent publication of the entire human genome, our knowledge of exactly what our genes say has increased immeasurably. This, however, is only a small part of the story. In order for the chromosomal genes to function correctly, a complex cellular machinery must rewrite (or transcribe) the genetic instructions of the DNA into a temporary messenger molecule, messenger RNA (mRNA), rearrange (or splice) this message into a readable format and then produce a protein that accurately represents the DNA code. It is these protein molecules that are the functional result of the genetic information. This whole process is termed 'gene expression'. Both transcription and splicing of the mRNA message are carried out in the nucleus. These events must be performed accurately and efficiently in a minute volume already full of highly packaged DNA. An ever-increasing number of sub-nuclear structures have been described, from the nucleolus (first described in 1835) to newly discovered 'paraspeckles' and 'clastosomes'. In fact, as increasing numbers of molecular probes become available, so the complexity of nuclear structure appears to expand. The functions of some of these structures are currently unknown. Those whose functions are, at least partly, understood play roles in gene expression. Interestingly, alterations in nuclear structure are associated with human diseases such as spinal muscular atrophy and promyelocytic leukaemia, suggesting that the control of nuclear organization may be vital to health. The dynamic nature of the structure of the mammalian nucleus has come under increasing scrutiny over the past few years. This has largely been driven by advances in microscopy as well as the advent of in vivo labelling techniques for sub-nuclear structures. It is now possible, using a protein originally isolated from jellyfish, to visualize sub-nuclear structures in living cultured cells. Together with three-dimensional time-lapse microscopy and an ever-expanding range of photo-bleaching techniques, this technology allows us to ask detailed questions about movements of sub-nuclear structures themselves and of the proteins contained within them. It has recently become clear that sub-nuclear structures are capable of moving within the nucleus and of physically interacting with each other. It is also now known that there is a constant flux of molecules into and out of these mobile structures as well as exchange of molecules between them, rather like passengers travelling on the London Underground. The challenge for the future is to relate dynamic events at the microscopic and molecular levels back to the organism as a whole. Only by understanding how the information encoded on genes is accurately expressed at the right time and in the right place can we really take advantage of the knowledge currently available to us.

Animals↗
PubMed2003

Cajal body proteins SMN and Coilin show differential dynamic behaviour in vivo.

Analysis of stable cell lines expressing fluorescently tagged survival of motor neurons protein (SMN) and coilin shows striking differences in their dynamic behaviour, both in the nucleus and during mitosis. Cajal bodies labelled with either FP-SMN or FP-coilin show similar behaviour and frequency of movements. However, fluorescence recovery after photobleaching (FRAP) studies show that SMN returns approximately 50-fold more slowly to Cajal bodies than does coilin. Time-lapse studies on cells progressing from prophase through to G1 show further differences between SMN and coilin, both in their localisation in telophase and in the timing of their re-entry into daughter nuclei. The data reveal similarities between Cajal bodies and nucleoli in their behaviour during mitosis. This in vivo study indicates that SMN and coilin interact differentially with Cajal bodies and reveals parallels in the pathway for reassembly of nucleoli and Cajal bodies following mitosis.

Autoantigens↗
PubMed2003

Protein phosphatase 4 interacts with the Survival of Motor Neurons complex and enhances the temporal localisation of snRNPs.

Protein phosphatase 4 (PPP4) is a ubiquitous essential protein serine/threonine phosphatase found in higher eukaryotes. Coordinate variation of the levels of the catalytic subunit (PPP4c) and the regulatory subunit (R2) suggests that PPP4c and R2 form a heterodimeric core to which other regulatory subunits bind. Two proteins that specifically co-purify with Flag-epitope-tagged R2 expressed in HEK-293 cells were identified as Gemin3 and Gemin4. These two proteins have been identified previously as components of the Survival of Motor Neurons (SMN) protein complex, which is functionally defective in the hereditary disorder spinal muscular atrophy. Immuno-sedimentation of the epitope-tagged SMN protein complex from HeLa cells expressing CFP-SMN showed that the SMN protein interacts, as previously reported, with Gemin2 (SIP1), Gemin3 and Gemin4 and in addition associates with PPP4c. The SMN complex has been implicated in the assembly and maturation of small nuclear ribonucleoproteins (snRNPs). Expression of GFP-R2-PPP4c in HeLa cells enhances the temporal localisation of newly formed snRNPs, which is consistent with an association of R2-PPP4c with the SMN protein complex.

Blotting, Northern↗