A cancer-associated mutation inactivates a region of the high-mobility group protein HMG20b essential for cytokinesis

Defects in the completion of cell division by cytokinesis have long been proposed to foster carcinogenesis by engendering chromosome instability, but few tumor suppressor mechanisms controlling this process have so far been identified. Here, we identify a carboxyl (C)-terminal region of the high-mobility group protein HMG20b that is essential for cytokinesis, and report that it is inactivated by a cancer-associated mutation. We find that a C-terminal region of HMG20b spanning residues 173–317 is necessary and sufficient not only for its localization to cytokinetic structures, but also for its interaction with the tumor suppressor BRCA2, implicated in the abscission step of cytokinesis. Indeed, expression of this C-terminal HMG20b region suffices to restore cytokinesis in HMG20b-depleted cells. The non-conservative substitution of HMG20b residue Ala247 with Pro, reported in human lung cancer, disrupts these activities of HMG20b, impairing cytokinesis in a trans-dominant manner. Our findings provide fresh insight into the mechanism by which the HMG20b-BRCA2 complex controls mitotic cell division, and implicate heterozygous HMG20b mutations affecting cytokinesis regulation in the genesis of human cancers.     

Beyond linker histones and high mobility group proteins: global profiling of perchloric acid soluble proteins

Extraction with HClO(4) provides an easy method for efficient enrichment of both histone H1 and HMG proteins from a variety of tissues. Usually, the histone and the HMG proteins are the most abundant components of the extracts, however, other proteins have frequently been observed but only seldom studied in more detail. Here we describe a study aimed at global characterization of HClO(4) extractable proteins from breast cancer cell lines. We report identification of 150 unique proteins by liquid chromatography tandem mass spectrometry including almost all major histone H1 variants and canonical members of the HMG protein families. In the extracts, diverse proteins with HMG-like amino acid composition were identified and their post-translational modifications were mapped. Importantly, those include multiple proteins known or supposed to be related to cell proliferation and cancer. Since purification of these proteins as well as low abundant variants of histone and HMG proteins is difficult due to their metabolic instability, characterization of these proteins from crude extracts can facilitate studies aimed at better understanding of their function.     

Insect proteins homologous to mammalian high mobility group protein 1. Characterization and DNA-binding properties.

Two chromosomal high mobility group (HMG) proteins from larvae of Chironomus thummi (Diptera) and from an epithelial cell line of Chironomus tentans were purified to homogeneity and chemically characterized. cDNA clones encoding these proteins were isolated from an expression library using an immunoscreening approach and were sequenced. The deduced amino acid sequences revealed their homology to HMG protein 1 of vertebrates. These insect proteins have therefore been designated cHMG1a and cHMG1b. They have a molecular mass of 12,915 and 12,019 kDa, respectively, and preferentially bind to AT-rich DNA. Indirect immunofluorescence microscopy with a polyclonal antibody showed the presence of cHMG1a and cHMG1b in condensed chromomeres but not in puffs, nucleoli, and cytoplasm. The cHMG1a and cHMG1b genes were both localized to a single band in region 14 of chromosome 1 of C. tentans and appear to be single copy genes. An immunologically related protein was purified from Drosophila melanogaster Kc cells. Its size and amino acid composition indicate that it is an HMG1 of D. melanogaster. On the other hand, our antibody did not recognize calf HMG1. The identification and characterization of HMG1 proteins in insects with polytene chromosomes opens new possibilities for studying function(s) of this group of chromosomal proteins.     

The effect of the acidic tail on the DNA-binding properties of the HMG1,2 class of proteins: insights from tail switching and tail removal

The high-mobility group (HMG) proteins HMG1, HMG2 and HMG2a are relatively abundant vertebrate DNA-binding and bending proteins that bind with structure specificity, rather than sequence specificity, and appear to play an architectural role in the assembly of nucleoprotein complexes. They have two homologous "HMG-box" DNA-binding domains (which show about 80 % homology) connected by a short basic linker to an acidic carboxy-terminal tail that differs in length between HMG1 and 2. To gain insights into the role of the acidic tail, we examined the DNA-binding properties of HMG1, HMG2b and HMG2a from chicken erythrocytes (corresponding to HMG1, HMG2 and HMG2a in other vertebrates). HMG1, with the longest acidic tail, is less effective than HMG2a and 2b (at a given molar input ratio) in supercoiling relaxed, closed circular DNA, in inducing ligase-mediated circularisation of an 88 bp DNA fragment, and in binding to four-way DNA junctions in a gel-shift assay. Removal of the acidic tail increases the affinity of the HMG boxes for DNA and largely abolishes the differences between the three species. Switching the acidic tail of HMG1 for that of HMG2a or 2b gives hybrid proteins with essentially the same DNA-binding properties as HMG2a, 2b. The length (and possibly sequence) of the acidic tail thus appears to be the dominant factor in mediating the differences in properties between HMG1, 2a and 2b and finely tunes the rather similar DNA-binding properties of the tandem HMG boxes, presumably to fulfill different cellular roles. The tail is essential for structure-selective DNA-binding of the HMG boxes to DNA minicircles in the presence of equimolar linear DNA, and has little effect on the affinity for this already highly distorted DNA ligand, in contrast to binding to linear and four-way junction DNA.     

Purification and postsynthetic modifications of Friend erythroleukemic cell high mobility group protein HMG-I

We have previously detected and purified a Friend erythroleukemic mouse cell nonhistone chromatin protein having extraction and acid-solubility properties like the low molecular weight "high mobility group" (HMG) nuclear proteins. We show here that the electrophoretic properties and the amino acid composition of this mouse cell "HMG-like" protein is comparable to those of the HMG-I proteins isolated from human HeLa S3 cells, African green monkey cells, Ehrlich ascites mouse cells, and rat fibroblast cells. Therefore, we have also designated the Friend erythroleukemic mouse cell protein as HMG-I. In common with the other HMG proteins the Friend cell HMG-I protein can undergo a variety of post-translational biochemical modifications including acetylation, ADP-ribosylation, glycosylation, and phosphorylation. Surprisingly, in the course of these studies we found that in vivo radiolabeling experiments revealed that only two minor HMG-14 subspecies (and/or possibly a minor HMG-I subspecies) are phosphorylated whereas HMG-1, -2, -17, and the major HMG-14 are not heavily phosphorylated.     

高迁移率族蛋白成员HMG20a/b的研究进展

高迁移率族(HMG)蛋白是一类广泛存在的非组蛋白型染色体蛋白,能通过诱导染色质结构的变化影响DNA表达。HMG20a和HMG20b是一对高度同源的HMG家族蛋白,均含有一个结构保守的HMG-box结构域和一个coiled-coil结构域,在生物体内广泛表达。HMG20a/b在细胞核内参与组蛋白去甲基酶复合物LSD1-Co REST的形成及一系列与细胞分裂分化相关的生理进程,如神经细胞核红细胞的分化、细胞质分裂以及EMT过程。研究发现,HMG20a/b一些功能的发挥是通过LSD1-Co REST复合物来实现的;在神经分化过程中,HMG20a、HMG20b具有相互拮抗的作用;而HMG20a促进EMT过程反映它很可能是一个促癌因子。本文对HMG20a/b的结构和体内分布及生物学功能进行综述。     

Comparative studies on microinjected high-mobility-group chromosomal proteins, HMG1 and HMG2

The nonhistone chromosomal proteins, HMG1 and HMG2, were iodinated and introduced into HeLa cells, bovine fibroblasts, or mouse 3T3 cells by erythrocyte-mediated microinjection. Autoradiographic analysis of injected cells fixed with glutaraldehyde consistently showed both molecules concentrated within nuclei. Fixation with methanol, on the other hand, resulted in some leakage of the microinjected proteins from the nuclei so that more autoradiographic grains appeared over the cytoplasm or outside the cells. Both injected and endogenous HMG1 and HMG2 partitioned unexpectedly upon fractionation of bovine fibroblasts, HeLa, or 3T3 cells, appearing in the cytoplasmic fractions. However, in calf thymus, HMG1 and HMG2 molecules appeared in the 0.35 M NaCl extract of isolated nuclei, as expected. These observations show that the binding of HMG1 and HMG2 to chromatin differs among cell types or that other tissue-specific components can influence their binding. Coinjection of [125I]HMG1 and [131I]HMG2 into HeLa cells revealed that the two molecules display virtually equivalent distributions upon cell fractionation, identical stability, identical intracellular distributions, and equal rates of equilibration between nuclei. In addition, HMG1 and HMG2 did not differ in their partitioning upon fractionation nor in their stability in growing vs. nongrowing 3T3 cells. Thus, we have not detected any significant differences in the intracellular behavior of HMG1 and HMG2 after microinjection into human, bovine, or murine cells.     

Fractionation by high-performance liquid chromatography of the low-molecular-mass high-mobility-group (HMG) chromosomal proteins present in proliferating rat cells and an investigation of the HMG proteins present in virus transformed cells

The low-molecular-mass high-mobility-group (HMG) proteins from young rat thymus nuclei were fractionated by high-performance liquid chromatography. Two proteins analogous to calf HMG14 and HMG17 were found together with a third major component HMGI similar to that found in HeLa cells [Lund et al. (1983) FEBS Lett. 152, 163-167]. HMGI has as amino acid composition similar to but distinct from HMG14 and HMG17. The three proteins form a family of proteins with HMG14 having an amino acid composition intermediate between HMG17 and HMGI. HMGI is present in proliferating fibroblasts and embryos but is present in very low levels in rat liver, a non-dividing tissue, supporting the notion that HMGI is required for proliferating cells. Fibroblasts transformed with avian sarcoma virus have high levels of HMGI and an additional band HMGI' but the presence of HMGI and HMGI' is not dependent on a functional src gene product.     

Drug modulation of chromosomal protein subtypes during specific phases of the submaxillary cell cycle

The H1 subtype proteins a, b, c, d, e, and 1 degree and the high mobility group (HMG) proteins 14 and 17 were extracted from submaxillary gland (SMG) nuclei treated in vivo and sorted from specific phases of the cell cycle, and analyzed by gel electrophoresis. Significant differences in these proteins were noted between quiescent (GO) phase nuclei and proliferating nuclei in both stained and autoradiographic gels. Stimulation of the SMG into cell division with DL-isoproterenol-HCl alone or in conjunction with sodium phenobarbital (PB) provided a system for the analysis of drug effects in stained, 3H lysine, and 32P pulse-labeled nuclear proteins obtained from different phases of the cell cycle.     

Phosphorylation of high mobility group 14 protein by cyclic nucleotide-dependent protein kinases

Chromosomal high mobility group (HMG) proteins have been examined as substrates for cGMP-dependent and cAMP-dependent protein kinases. Of the four HMG proteins only HMG 14 contained a major high affinity site which could be phosphorylated by both enzymes, preferentially by cGMP-dependent protein kinase. One mol of 32P was incorporated/mol of HMG 14. Kinetic analysis revealed apparent Km and Vmax of 40.5 microM and 14.7 mumol/min/mg, respectively, for cGMP-dependent protein kinase, and 123 microM and 11.1 mumol/min/mg, respectively, for cAMP-dependent protein kinase. Tryptic maps of 32P-labeled phosphopeptides of HMG 14 demonstrated phosphorylation of the same site by both enzymes. The tryptic fragment containing the major phosphorylation site was identified by amino acid composition and sequence as HMG 14 (residues 4-13): H-Lys-Val-Ser(P)-Ser-Ala-Glu-Gly-Ala-Ala-Lys-OH. HMG 14 and HMG 17 also contained minor sites which could be phosphorylated by cGMP-dependent protein kinase. Tryptic phosphopeptides mapping suggested that the same minor site was phosphorylated on both HMG 14 and 17. On the basis of amino acid composition, the tryptic peptides carrying the minor phosphorylation sites were identified as H-Leu-Ser(P)-Ala-Lys representing residues 23-26 and 27-30 of HMG 14 and HMG 17, respectively.     

High mobility group nonhistone chromosomal proteins of the developing sea urchin embryo

The high mobility group or HMG proteins are nonhistone chromosomal proteins that have been found in relatively high amounts in nuclei of many tissues. A number of studies have shown that some of these proteins are preferentially associated with actively transcribed regions of the genome and may play a role in maintaining these regions in an active state. In this study, we undertook an investigation of the high mobility group proteins from the sea urchin, Stronglyocentrotus purpuratus. Initially the putative sea urchin HMGs were extracted from isolated nuclei of hatching blastula-stage embryos with 5% perchloric acid (PCA). The major proteins in this extract were characterized according to their electrophoretic mobility, amino acid composition, and association with isolated deoxyribonucleoprotein particles. The results indicate there is only one "major" sea urchin HMG protein, termed P2 in this paper. An estimate of the amount of P2 in relation to the inner histones, however, was low compared to what has been found for other HMG proteins. Of the other major 5% PCA-extractable proteins, one was identified as the cleavage stage H1. Another protein apparently resulted from H3 contamination in the 5% PCA extract, and the fourth major protein did not have all the characteristics of an HMG. In particular, it was not found associated with nucleosomal particles. The HMG proteins from other developmental stages were then examined. Five percent PCA extracts of nuclei from unfertilized eggs, 2-cell, 16-cell, hatching blastula, gastrula, and pluteus stages were analyzed on SDS- and acetic acid-urea gels. This analysis indicated that P2 exists in two different forms differing slightly in charge. The less basic form was found in the egg, 2-cell and 16-cell extracts. At the hatching blastula stage, both forms were present and by pluteus stage, the more basic form predominated. It appears that P2 is undergoing a developmental change from a less to more basic form. The presence of P2 in the 5% PCA extract of egg nuclei is proof that P2 does not initially appear sometime during embryogenesis but is already in the egg nucleus prior to fertilization.     

Extraction and partial characterization of non-histone nuclear proteins of Schistosoma mansoni.

A pool of nuclear proteins from adult worms of Schistosoma mansoni was analyzed for amino acid composition and found to be compatible with high mobility group (HMG) proteins. One of the schistosome HMG proteins was identified as HMG 2 by one-dimensional and two-dimensional PAGE. Stage-specific differences in the HMG-like protein composition were encountered when adult worms were compared to schistosomula, the larval form. Immobilization of the adult male and female nuclear proteins onto nitrocellulose, followed by hybridization against 32P-F-10, a schistosome sex specific gene encoding a major egg shell protein, revealed distinct banding patterns. On the other hand, a synthetic oligonucleotide, derived from the 3' untranslated end of the F-10 gene and possibly containing one regulatory element of the gene, bound mainly to male low MW proteins.     

Frequent Detection of Hepatitis C Virus Subtype 3a (HCV-3a) Isolates in Thailand by PCR Using Subtype-Specific Primers

By means of a polymerase chain reaction (PCR) method using subtype-specific primers for hepatitis C virus (HCV) subtypes 1a, 1b, 2a, 2b and 3a, the prevalence of each subtype among HCV isolates in Chiang Mai, Thailand, was determined. HCV-3a appeared to be the most common subtype in blood donors, and was also frequently found in patients with liver disease. HCV-1b, but not HCV-2a or ?2b, was also commonly found in this area, while a considerable percentage of the total HCV isolates still remained unclassifiable by the above methods. Serotype analysis of the HCV isolates using C14-1 and C14-2 recombinant peptides revealed that HCV-3a was likely to carry an antigenic determinant(s) different from those of the major types 1 (HCV-1a and ?1b) and 2 (HCV-2a and ?2b).     

北京鸭HMGs和HP_1的分离纯化及其对细胞核体外转录的影响

本文用改进的CM-Sephadex C-25柱层析的方法,纯化了HMG各组份及HP_1。在北京鸭嗜多染红细胞核体外转录体系中测定了HP_1、HMGs及HP_1+HMGs对转录活性的影响,实验结果表明,单独的HMGs和单独的HP_1对转录活性均无显著性的影响,而HP_1+HMGs对转录活性有明显的抑制作用。