HMGA Proteins: Isolation, Biochemical Modifications, and Nucleosome Interactions
Introduction
The mammalian HMGA family of high mobility group (HMG) nonhistone proteins [formerly called HMG-I(Y) proteins1] has been referred to as a “hub” of nuclear function because of its intimate involvement in a wide variety of biology processes.2 An extensive literature suggests that HMGA proteins act as architectural transcription factors that function by recognizing and modulating the structure of both DNA and chromatin substrates and, as a consequence, have the ability to participate in a diverse array of nuclear events ranging from DNA replication and repair to the regulation of gene transcription and mRNA splicing and the control of integration of retroviruses into the genome (reviewed in Ref. 3). HMGA proteins have also attracted considerable medical interest because their aberrant expression or overexpression has been demonstrated to lead to neoplastic transformation of cells and to promote metastatic progression of cancers (reviewed in Refs. 3, 4, 5, 6).
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HMGA Proteins Exhibit Unusual Properties
In both mice and humans, the principal members of the HMGA protein family (i.e., HMGA1a, HMGA1b, and HMG2) are produced by translation of alternatively spliced mRNA transcripts encoded by two different genes, HMGA1 and HMGA2 (reviewed in Refs. 6, 7). HMGA proteins share a number of common biochemical, biophysical, and biological properties that distinguish them from other nuclear proteins. The major HMGA protein species are small (∼10.6–12 kDa), have unusually high concentrations of basic,
Production and Purification of Recombinant HMGA Proteins
Wild-type, mutant, and truncated forms of recombinant HMGA proteins produced in either bacteria or insect cells have been widely used in studies to elucidate their in vitro biochemical, biophysical, and biological characteristics. Unfortunately, however, it is often difficult to isolate appreciable quantities of recombinant HMGA proteins because their levels of production in many host cell expression systems are frequently low. Among the factors that contribute to this low level of expression
Isolation and Purification of HMGA Proteins from Mammalian Cells and Tissues
Two different methods have been widely used to extract HMGA proteins from the nuclei and chromatin of mammalian cells. Either they can be eluted by extraction with dilute acids, as described above for recombinant proteins, or they can be extracted with a buffered solution containing 0.3–0.4 M NaCl.27 There are advantages and disadvantages to both methods. Although salt extraction is mild and allows for relatively efficient recovery of native, nondenatured HMGA proteins, it results in the
In Vivo Biochemical Modifications of HMGA Proteins
HMGA proteins isolated from living cells exhibit complex patterns of postsynthetic phosphorylations, acetylations, and methylations, making them among the most highly modified proteins in the mammalian nucleus (reviewed in Refs. 2, 3). These modifications are dynamic and rapidly change as a function of the cell cycle,30, 31 the state of cellular differentiation,32 in response to intra- and extracellular signaling events,32 and during the activation of apoptotic cell death.33, 34
The diagram in
Determination of In Vivo Biochemical Modifications by Mass Spectrometry
Although a variety of procedures have been employed to determine the sites of individual types of in vivo biochemical modifications on HMGA proteins, mass spectrometry (MS) techniques have proved to be the most efficient and facile methods for analyzing the complex patterns of multiple modifications that are simultaneously present on these proteins in living cells. Matrix-assisted laser desorption⧸ionization time-of-flight (MALDI/TOF) MS has been successfully used in our laboratory to analyze
HMGA Proteins Bind to Nucleosome Core Particles Both In Vitro and In Vivo
Although HMGA proteins have received a considerable amount of attention because of their active role in regulating the transcription of more than 40 different eukaryotic and viral genes by participating in the formation of stereospecific, multiprotein complexes called “enhanceosomes” on their promoter⧸enhancer regions (reviewed in Refs. 3, 48), these proteins have long been recognized as being important participants in many other nuclear processes. HMGA proteins are integral components of both
HMGA1 Binding to Nucleosomes Is Orientation Specific
Interaction of HMGA1 proteins with nucleosomes not only induces localized changes in the rotational setting of DNA on core particles,13 it also binds to A⧸T-rich stretches of DNA on the nucleosome surface in a orientation-specific manner.53 HMGA1 proteins participate in the transcriptional induction of the gene encoding the α subunit of the human interleukin 2 receptor (IL-2Rα) in stimulated T lymphocytes by controlling the formation of an enhanceosome on the gene's 5′ proximal promoter region.
Isolation, Mutagenesis, and Radiolabeling of DNA Fragments
A cloned 581-bp BamHI–PstI restriction fragment encompassing nucleotides −472 to +109 of the human IL-2Rα gene and its 5′ proximal promoter region57 serves as the starting material for isolating subfragments of the promoter containing the PRRII element for use in in vitro chromatin reconstitution experiments. Standard polymerase chain reaction (PCR) techniques22 are used to amplify a 277-bp promoter fragment that encompasses PRRII and its flanking regions, using the following PCR primer pair:
A⧸T-Hook Peptide Motif Binds Nucleosome Core Particles
Studies of cocomplexes of HMGA1 proteins with synthetic DNA substrates, using solution nuclear magnetic resonance (NMR) techniques, revealed that A⧸T-hook peptides that interact with the minor groove of A⧸T-rich sequences of DNA do so in a direction-specific manner,8 raising a question concerning whether the A⧸T-hook regions of these proteins are also responsible for mediating the directional interactions with nucleosome core particles. Of even greater biological importance is determining
A⧸T Hooks Are Essential Components of Many Chromatin-Remodeling Complexes
The observation that HMGA proteins bind to nucleosome core particles via their A⧸T-hook motifs and, thereby, induce localized changes in the structure of DNA on the surface of core particles has taken on unanticipated significance. It is now becoming apparent that A⧸T hook-containing proteins are essential components of many of the multi-protein, ATP-dependent chromatin-remodeling complexes (or “machines”; CRMs) found in yeast, Drosophila, and mammalian cells (reviewed in Refs. 2, 3). For
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HMGA-targeted phosphorothioate DNA aptamers increase sensitivity to gemcitabine chemotherapy in human pancreatic cancer cell lines
2012, Cancer LettersCitation Excerpt :E. coli expressing HMGA1b was cultured at 37 °C to an OD600 measurement of 0.8–1.0. Protein expression was induced by the addition of 1 mM IPTG and shaking at 37 °C for 4–6 h. HMGA1 was purified by trichloroacetic acid precipitation as described previously [29]. Overexpressed HMGA1b was further purified with a Sephadex G-25 column in H2O and lyophilized.
Collective mass spectrometry approaches reveal broad and combinatorial modification of high mobility group protein a1a
2010, Journal of the American Society for Mass SpectrometryCitation Excerpt :HeLa S3 cells were grown and harvested as described by Thomas et al. [36], while nuclei were isolated as described by Garcia et al. [37], and HMG proteins were acid extracted from HeLa cells as described by Reeves [38].
High mobility group proteins and their post-translational modifications
2008, Biochimica et Biophysica Acta - Proteins and ProteomicsA Quantitative Study on the In Vitro and In Vivo Acetylation of High Mobility Group A1 Proteins
2007, Journal of the American Society for Mass SpectrometryCitation Excerpt :Full-length recombinant human HMGA1a and HMGA1b proteins were overexpressed in E. coli BL21 DE3 pLysS cells (Invitrogen, Carlsbad, CA) followed by extraction with 5% perchloric acid (PCA) as reported previously [44, 45].