Publications by authors named "Michael L Mimmack"

10 Publications

  • Page 1 of 1

The Impact of the C-Terminal Region on the Interaction of Topoisomerase II Alpha with Mitotic Chromatin.

Int J Mol Sci 2019 Mar 12;20(5). Epub 2019 Mar 12.

Department of Genetics, University of Cambridge, Downing St, Cambridge CB2 3EH, UK.

Type II topoisomerase enzymes are essential for resolving DNA topology problems arising through various aspects of DNA metabolism. In vertebrates two isoforms are present, one of which (TOP2A) accumulates on chromatin during mitosis. Moreover, TOP2A targets the mitotic centromere during prophase, persisting there until anaphase onset. It is the catalytically-dispensable C-terminal domain of TOP2 that is crucial in determining this isoform-specific behaviour. In this study we show that, in addition to the recently identified chromatin tether domain, several other features of the alpha-C-Terminal Domain (CTD). influence the mitotic localisation of TOP2A. Lysine 1240 is a major SUMOylation target in cycling human cells and the efficiency of this modification appears to be influenced by T1244 and S1247 phosphorylation. Replacement of K1240 by arginine results in fewer cells displaying centromeric TOP2A accumulation during prometaphase-metaphase. The same phenotype is displayed by cells expressing TOP2A in which either of the mitotic phosphorylation sites S1213 or S1247 has been substituted by alanine. Conversely, constitutive modification of TOP2A by fusion to SUMO2 exerts the opposite effect. FRAP analysis of protein mobility indicates that post-translational modification of TOP2A can influence the enzyme's residence time on mitotic chromatin, as well as its subcellular localisation.
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http://dx.doi.org/10.3390/ijms20051238DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6429393PMC
March 2019

Dual binding motifs underpin the hierarchical association of perilipins1-3 with lipid droplets.

Mol Biol Cell 2019 03 16;30(5):703-716. Epub 2019 Jan 16.

Laboratoire de Physique de l'Ecole Normale Supérieure, ENS, Université PSL, CNRS, Sorbonne Université, Université Paris-Diderot, Sorbonne Paris Cité, Paris, France.

Lipid droplets (LDs) in all eukaryotic cells are coated with at least one of the perilipin (Plin) family of proteins. They all regulate key intracellular lipases but do so to significantly different extents. Where more than one Plin is expressed in a cell, they associate with LDs in a hierarchical manner. In vivo, this means that lipid flux control in a particular cell or tissue type is heavily influenced by the specific Plins present on its LDs. Despite their early discovery, exactly how Plins target LDs and why they displace each other in a "hierarchical" manner remains unclear. They all share an amino-terminal 11-mer repeat (11mr) amphipathic region suggested to be involved in LD targeting. Here, we show that, in vivo, this domain functions as a primary highly reversible LD targeting motif in Plin1-3, and, in vitro, we document reversible and competitive binding between a wild-type purified Plin1 11mr peptide and a mutant with reduced binding affinity to both "naked" and phospholipid-coated oil-water interfaces. We also present data suggesting that a second carboxy-terminal 4-helix bundle domain stabilizes LD binding in Plin1 more effectively than in Plin2, whereas it weakens binding in Plin3. These findings suggest that dual amphipathic helical regions mediate LD targeting and underpin the hierarchical binding of Plin1-3 to LDs.
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http://dx.doi.org/10.1091/mbc.E18-08-0534DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6589688PMC
March 2019

Conserved Amphipathic Helices Mediate Lipid Droplet Targeting of Perilipins 1-3.

J Biol Chem 2016 Mar 7;291(13):6664-78. Epub 2016 Jan 7.

From the University of Cambridge Metabolic Research Laboratories, Wellcome Trust-Medical Research Council Institute of Metabolic Science, Cambridge CB2 0QQ, United Kingdom,

Perilipins (PLINs) play a key role in energy storage by orchestrating the activity of lipases on the surface of lipid droplets. Failure of this activity results in severe metabolic disease in humans. Unlike all other lipid droplet-associated proteins, PLINs localize almost exclusively to the phospholipid monolayer surrounding the droplet. To understand how they sense and associate with the unique topology of the droplet surface, we studied the localization of human PLINs inSaccharomyces cerevisiae,demonstrating that the targeting mechanism is highly conserved and that 11-mer repeat regions are sufficient for droplet targeting. Mutations designed to disrupt folding of this region into amphipathic helices (AHs) significantly decreased lipid droplet targetingin vivoandin vitro Finally, we demonstrated a substantial increase in the helicity of this region in the presence of detergent micelles, which was prevented by an AH-disrupting missense mutation. We conclude that highly conserved 11-mer repeat regions of PLINs target lipid droplets by folding into AHs on the droplet surface, thus enabling PLINs to regulate the interface between the hydrophobic lipid core and its surrounding hydrophilic environment.
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http://dx.doi.org/10.1074/jbc.M115.691048DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4807253PMC
March 2016

The α isoform of topoisomerase II is required for hypercompaction of mitotic chromosomes in human cells.

Nucleic Acids Res 2014 Apr 29;42(7):4414-26. Epub 2014 Jan 29.

Department of Genetics, University of Cambridge, Downing St, Cambridge CB2 3EH, UK and Centre for Haematology, Faculty of Medicine, Imperial College London, Hammersmith Hospital Campus, Du Cane Rd, London W12 0NN, UK.

As proliferating cells transit from interphase into M-phase, chromatin undergoes extensive reorganization, and topoisomerase (topo) IIα, the major isoform of this enzyme present in cycling vertebrate cells, plays a key role in this process. In this study, a human cell line conditional null mutant for topo IIα and a derivative expressing an auxin-inducible degron (AID)-tagged version of the protein have been used to distinguish real mitotic chromosome functions of topo IIα from its more general role in DNA metabolism and to investigate whether topo IIβ makes any contribution to mitotic chromosome formation. We show that topo IIβ does contribute, with endogenous levels being sufficient for the initial stages of axial shortening. However, a significant effect of topo IIα depletion, seen with or without the co-depletion of topo IIβ, is the failure of chromosomes to hypercompact when delayed in M-phase. This requires much higher levels of topo II protein and is impaired by drugs or mutations that affect enzyme activity. A prolonged delay at the G2/M border results in hyperefficient axial shortening, a process that is topo IIα-dependent. Rapid depletion of topo IIα has allowed us to show that its function during late G2 and M-phase is truly required for shaping mitotic chromosomes.
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http://dx.doi.org/10.1093/nar/gku076DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3985649PMC
April 2014

Genome-wide association study of CNVs in 16,000 cases of eight common diseases and 3,000 shared controls.

Nature 2010 Apr;464(7289):713-20

Copy number variants (CNVs) account for a major proportion of human genetic polymorphism and have been predicted to have an important role in genetic susceptibility to common disease. To address this we undertook a large, direct genome-wide study of association between CNVs and eight common human diseases. Using a purpose-designed array we typed approximately 19,000 individuals into distinct copy-number classes at 3,432 polymorphic CNVs, including an estimated approximately 50% of all common CNVs larger than 500 base pairs. We identified several biological artefacts that lead to false-positive associations, including systematic CNV differences between DNAs derived from blood and cell lines. Association testing and follow-up replication analyses confirmed three loci where CNVs were associated with disease-IRGM for Crohn's disease, HLA for Crohn's disease, rheumatoid arthritis and type 1 diabetes, and TSPAN8 for type 2 diabetes-although in each case the locus had previously been identified in single nucleotide polymorphism (SNP)-based studies, reflecting our observation that most common CNVs that are well-typed on our array are well tagged by SNPs and so have been indirectly explored through SNP studies. We conclude that common CNVs that can be typed on existing platforms are unlikely to contribute greatly to the genetic basis of common human diseases.
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http://dx.doi.org/10.1038/nature08979DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2892339PMC
April 2010

Affinity ligands for immunoglobulins based on the multicomponent Ugi reaction.

J Chromatogr B Analyt Technol Biomed Life Sci 2009 May 16;877(14-15):1440-52. Epub 2009 Mar 16.

Department of Chemical Engineering and Biotechnology, Institute of Biotechnology, University of Cambridge, Cambridge, UK.

This report describes a novel use of the four-component Ugi reaction to generate a solid-phase library suitable for the purification of immunoglobulins and their fragments by affinity chromatography. An aldehyde-functionalised Sepharose solid-support constituted one component in the four-component reaction, whereas the other three components (a carboxylic acid, a primary or secondary amine and an isonitrile) were varied in a combinatorial fashion to generate a tri-substituted peptoidal scaffold structure which provides a degree of rigidity and functionality suitable for rational investigation of immunoglobulin binding. The Ugi ligand library was initially screened chromatographically against whole human IgG and its fragments (Fc and Fab) to yield a Fab-specific lead ligand based on its ability to bind Fab differentially over Fc. Preparative chromatography of IgG from human serum showed 100% of IgG was adsorbed from the 20mg/ml crude stock and subsequently eluted with a purity of 81.0% as determined by SDS-PAGE analysis under non-optimised conditions. High purity Fab and IgG isolation was achieved from both yeast and E. coli host cell proteins according to silver-stained SDS-PAGE lane densitometry. The ligand density and spacer-arm chemistry of the immobilised ligand was optimised to define an affinity adsorbent which binds 73.06 mg IgG/ml moist gel (dynamic binding capacity at 10% breakthrough) and a static binding capacity of 16.1+/-0.25mg Fab/ml moist resin displaying an affinity constant K(d)=(2.6+/-0.3)x10(-6)M. The lead candidate was modelled in silico and docked into a human Fab fragment (PDB: 1AQK) to suggest a putative binding interface to the constant CH(1)-CL Fab terminal through six defined hydrogen bond interactions together with putative hydrophobic interactions.
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http://dx.doi.org/10.1016/j.jchromb.2009.03.010DOI Listing
May 2009

Further evidence for altered myelin biosynthesis and glutamatergic dysfunction in schizophrenia.

Int J Neuropsychopharmacol 2007 Aug 12;10(4):557-63. Epub 2007 Feb 12.

Institute of Biotechnology, Tennis Court Road, Cambridge, UK.

Recent studies have provided evidence for neuronal and oligodendrocyte-related abnormalities being associated with schizophrenia. However, the functional interplay and causal relationship between these two abnormalities is poorly understood. In this report, we provide data that identify myelin and fatty-acid biosynthesis dysfunction in schizophrenia based on post-mortem brain studies (prefrontal cortex) utilizing parallel metabolic and transcriptomics investigations. We detected a significant up-regulation of N-acetylaspartate (NAA) by HPLC analysis. Microarray and Q-PCR investigations revealed mRNA abnormalities for several enzymes involved in NAA metabolism. Additionally, glutamatergic neurotransmission components were also found to be affected. Our results suggest that, apart from the previously reported alterations in myelin-related protein synthesis, myelin synthesis itself may be directly affected in schizophrenia as indicated by changes in key enzymes involved in NAA metabolism. A decrease in NAA catabolism in oligodendrocytes would severely reduce acetate levels required to produce myelin lipids and may subsequently affect glutamatergic neurotransmission.
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http://dx.doi.org/10.1017/S1461145706007334DOI Listing
August 2007

Quantitative polymerase chain reaction: validation of microarray results from postmortem brain studies.

Biol Psychiatry 2004 Feb;55(4):337-45

Department of Neurobiology, The Babraham Institute, Babraham, Cambridge CB2 4AT, United Kingdom.

Quantitative polymerase chain reaction (Q-PCR) is now considered the "technique of choice" for validating gene expression changes identified with ribonucleic acid-based expression profiling technologies (especially micro- and macroarray techniques). The identification of altered gene expression profiles with microarrays is best viewed as the first step in the determination of potential disease-associated genes; however, the false-positive rate can be high, particularly with small sample sets and in view of the typically small differences observed in brain expression studies. Quantitative PCR is a rapid and highly sensitive technique for accurate quantification of microarray results; however, careful consideration of experimental design, quality of primer/probe design, internal standards, and normalization procedures are pivotal, particularly when the work involves postmortem tissue.
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http://dx.doi.org/10.1016/j.biopsych.2003.09.007DOI Listing
February 2004

Oligodendrocyte dysfunction in schizophrenia and bipolar disorder.

Lancet 2003 Sep;362(9386):798-805

Department of Neurobiology, Babraham Institute, Cambridge, UK.

Background: Results of array studies have suggested abnormalities in expression of lipid and myelin-related genes in schizophrenia. Here, we investigated oligodendrocyte-specific and myelination-associated gene expression in schizophrenia and bipolar affective disorder.

Methods: We used samples from the Stanley brain collection, consisting of 15 schizophrenia, 15 bipolar affective disorder, and 15 control brains. Indexing-based differential display PCR was done to screen for differences in gene expression in schizophrenia patients versus controls. Results were cross-validated with quantitative PCR, which was also used to investigate expression profiles of 16 other oligodendrocyte and myelin genes in schizophrenia and bipolar disorder. These genes were further investigated with an ongoing microarray analysis.

Findings: Results of differential display and quantitative PCR analysis showed a reduction of key oligodendrocyte-related and myelin-related genes in schizophrenia and bipolar patients; expression changes for both disorders showed a high degree of overlap. Microarray results of the same genes investigated by quantitative PCR correlated well overall.

Interpretation: Schizophrenia and bipolar brains showed downregulation of key oligodendrocyte and myelination genes, including transcription factors that regulate these genes, compared with control brains. These results lend support to and extend observations from other microarray investigations. Our study also showed similar expression changes to the schizophrenia group in bipolar brains, which thus lends support to the notion that the disorders share common causative and pathophysiological pathways.
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http://dx.doi.org/10.1016/S0140-6736(03)14289-4DOI Listing
September 2003

Gene expression analysis in schizophrenia: reproducible up-regulation of several members of the apolipoprotein L family located in a high-susceptibility locus for schizophrenia on chromosome 22.

Proc Natl Acad Sci U S A 2002 Apr;99(7):4680-5

Department of Neurobiology, Babraham Institute, Cambridge CB2 4AT, United Kingdom.

We screened a custom-made candidate gene cDNA array comprising 300 genes. Genes chosen have either been implicated in schizophrenia, make conceptual sense in the light of the current understanding of the disease, or are located on high-susceptibility chromosome locations. The array screen using prefrontal cortex tissue from 10 schizophrenia and 10 control brains revealed robust up-regulation of apolipoprotein L1 (apo L1) by 2.6-fold. The finding was cross-validated in a blinded quantitative PCR study using prefrontal cortex tissue from the Stanley Foundation brain collection, Bethesda, MD. This collection consists of 15 schizophrenia, 15 bipolar disorder, 15 major depression, and 15 control individuals, all 60 brains being well-matched on conventional parameters, with antipsychotic drug exposure in the schizophrenia and bipolar disorder groups. Significant up-regulation of apo L1 gene expression in schizophrenia was confirmed. Using quantitative PCR, expression profiles of other members of the apo L family (apo L2-L6) were investigated, showing that apo L2 and L4 were highly significantly up-regulated in schizophrenia. Results were then confirmed in an independent set of 20 schizophrenia and 20 control brains from Japan and New Zealand. Apo L proteins belong to the group of high density lipoproteins, with all six apo L genes located in close proximity to each other on chromosome 22q12, a confirmed high-susceptibility locus for schizophrenia and close to the region associated with velocardiofacial syndrome that includes symptoms of schizophrenia.
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http://dx.doi.org/10.1073/pnas.032069099DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC123707PMC
April 2002