RESUMEN
Extracellular HMGB1 protein is known to induce inflammatory responses leading to an inflammatory storm. The outbreak of the Severe Acute Respiratory Syndrome COVID-19 due to the SARS-CoV-2 virus has resulted in a huge health concern worldwide. Recent data revealed that plasma/serum HMGB1 levels of patients suffering from inflammation-mediated disorders-such as COVID-19, cancer, and autoimmune disorders-correlate positively with disease severity and vice versa. A late release of HMGB1 in sepsis suggests the existence of a wide therapeutic window for treating sepsis. Rapid and accurate methods for the detection of HMGB1 levels in plasma/serum are, therefore, of great importance for monitoring the occurrence, treatment success, and survival prediction of patients with inflammation-mediated diseases. In this review, we briefly explain the role of HMGB1 in the cell, and particularly the involvement of extracellular HMGB1 (released from the cells) in inflammation-mediated diseases, with an emphasis on COVID-19. The current assays to measure HMGB1 levels in human plasma-Western blotting, ELISA, EMSA, and a new approach based on electrochemical immunosensors, including some of our preliminary results-are presented and thoroughly discussed.
Asunto(s)
COVID-19 , Proteína HMGB1 , Sepsis , Técnicas Biosensibles , COVID-19/sangre , COVID-19/diagnóstico , Proteína HMGB1/sangre , Humanos , Inmunoensayo , Pronóstico , SARS-CoV-2RESUMEN
HMGB1 and HMGB2 proteins are abundantly expressed in human embryonic stem cells (hESCs) and hESC-derived progenitor cells (neuroectodermal cells, hNECs), though their functional roles in pluripotency and the mechanisms underlying their differentiation in response to the anticancer drug etoposide remain to be elucidated. Here, we show that HMGB1 and/or HMGB2 knockdown (KD) by shRNA in hESCs did not affect the cell stemness/pluripotency regardless of etoposide treatments, while in hESC-derived neuroectodermal cells, treatment resulted in differential effects on cell survival and the generation of rosette structures. The objective of this work was to determine whether HMGB1/2 proteins could modulate the sensitivity of hESCs and hESC-derived progenitor cells (hNECs) to etoposide. We observed that HMGB1 KD knockdown (KD) and, to a lesser extent, HMGB2 KD enhanced the sensitivity of hESCs to etoposide. Enhanced accumulation of 53BP1 on telomeres was detected by confocal microscopy in both untreated and etoposide-treated HMGB1 KD hESCs and hNECs, indicating that the loss of HMGB1 could destabilize telomeres. On the other hand, decreased accumulation of 53BP1 on telomeres in etoposide-treated HMGB2 KD hESCs (but not in HMGB2 KD hNECs) suggested that the loss of HMGB2 promoted the stability of telomeres. Etoposide treatment of hESCs resulted in a significant enhancement of telomerase activity, with the highest increase observed in the HMGB2 KD cells. Interestingly, no changes in telomerase activity were found in etoposide-treated control hNECs, but HMGB2 KD (unlike HMGB1 KD) markedly decreased telomerase activity in these cells. Changes in telomerase activity in the etoposide-treated HMGB2 KD hESCs or hNECs coincided with the appearance of DNA damage markers and could already be observed before the onset of apoptosis. Collectively, we have demonstrated that HMGB1 or HMGB2 differentially modulate the impact of etoposide treatment on human embryonic stem cells and their progenitor cells, suggesting possible strategies for the enhancement of the efficacy of this anticancer drug.
Asunto(s)
Etopósido/farmacología , Proteína HMGB1/genética , Proteína HMGB2/genética , Neoplasias/tratamiento farmacológico , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Diferenciación Celular/genética , Regulación Neoplásica de la Expresión Génica/genética , Proteína HMGB1/antagonistas & inhibidores , Proteína HMGB2/antagonistas & inhibidores , Células Madre Embrionarias Humanas , Humanos , Neoplasias/genética , Neoplasias/patología , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/metabolismo , ARN Interferente Pequeño , Células Madre/efectos de los fármacos , Telomerasa/genéticaRESUMEN
High-mobility group box (HMGB)1 and HMGB2 proteins are the subject of intensive research because of their involvement in DNA replication, repair, transcription, differentiation, proliferation, cell signaling, inflammation, and tumor migration. Using inducible, stably transfected human embryonic stem cells (hESCs) capable of the short hairpin RNA-mediated knockdown (KD) of HMGB1 and HMGB2, we provide evidence that deregulation of HMGB1 or HMGB2 expression in hESCs and their differentiated derivatives (neuroectodermal cells) results in distinct modulation of telomere homeostasis. Whereas HMGB1 enhances telomerase activity, HMGB2 acts as a negative regulator of telomerase activity in the cell. Stimulation of telomerase activity in the HMGB2-deficient cells may be related to activation of the PI3K/protein kinase B/ glycogen synthase kinase-3ß/ß-catenin signaling pathways by HMGB1, augmented TERT/telomerase RNA subunit transcription, and possibly also because of changes in telomeric repeat-containing RNA (TERRA) and TERRA-polyA+ transcription. The impact of HMGB1/2 KD on telomerase transcriptional regulation observed in neuroectodermal cells is partially masked in hESCs by their pluripotent state. Our findings on differential roles of HMGB1 and HMGB2 proteins in regulation of telomerase activity may suggest another possible outcome of HMGB1 targeting in cells, which is currently a promising approach aiming at increasing the anticancer activity of cytotoxic agents.-Kucírek, M., Bagherpoor, A. J., Jaros, J., Hampl, A., Stros, M. HMGB2 is a negative regulator of telomerase activity in human embryonic stem and progenitor cells.
Asunto(s)
Proteína HMGB2/fisiología , Células Madre Embrionarias Humanas/enzimología , Células Madre/enzimología , Telomerasa/metabolismo , Diferenciación Celular , Proteína HMGB1/genética , Proteína HMGB2/genética , Células Madre Embrionarias Humanas/citología , Humanos , Células Madre/citología , Transcripción Genética , TransfecciónRESUMEN
HMGB1 is a chromatin-associated protein that has been implicated in many important biological processes such as transcription, recombination, DNA repair, and genome stability. These functions include the enhancement of binding of a number of transcription factors, including the tumor suppressor protein p53, to their specific DNA-binding sites. HMGB1 is composed of two highly conserved HMG boxes, linked to an intrinsically disordered acidic C-terminal tail. Previous reports have suggested that the ability of HMGB1 to bend DNA may explain the in vitro HMGB1-mediated increase in sequence-specific DNA binding by p53. The aim of this study was to reinvestigate the importance of HMGB1-induced DNA bending in relationship to the ability of the protein to promote the specific binding of p53 to short DNA duplexes in vitro, and to transactivate two major p53-regulated human genes: Mdm2 and p21/WAF1. Using a number of HMGB1 mutants, we report that the HMGB1-mediated increase in sequence-specific p53 binding to DNA duplexes in vitro depends very little on HMGB1-mediated DNA bending. The presence of the acidic C-terminal tail of HMGB1 and/or the oxidation of the protein can reduce the HMGB1-mediated p53 binding. Interestingly, the induction of transactivation of p53-responsive gene promoters by HMGB1 requires both the ability of the protein to bend DNA and the acidic C-terminal tail, and is promoter-specific. We propose that the efficient transactivation of p53-responsive gene promoters by HMGB1 depends on complex events, rather than solely on the promotion of p53 binding to its DNA cognate sites.
Asunto(s)
ADN/química , ADN/metabolismo , Proteína HMGB1/metabolismo , Conformación de Ácido Nucleico , Regiones Promotoras Genéticas , Activación Transcripcional/genética , Proteína p53 Supresora de Tumor/metabolismo , Línea Celular Tumoral , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Proteína HMGB1/química , Humanos , Proteínas Mutantes/metabolismo , Mutación/genética , Oxidación-Reducción , Unión Proteica , Dominios Proteicos , Proteínas Proto-Oncogénicas c-mdm2/genética , Proteínas Proto-Oncogénicas c-mdm2/metabolismoRESUMEN
HMGB1 and HMGB2 proteins have been implicated in numerous cellular processes, including proliferation, differentiation, apoptosis, and tumor growth. It is unknown whether they are involved in regulating the typical functions of pluripotent human embryonic stem cells (hESCs) and/or those of the differentiated derivatives of hESCs. Using inducible, stably transfected hESCs capable of shRNA-mediated knockdown of HMGB1 and HMGB2, we provide evidence that downregulation of HMGB1 and/or HMGB2 in undifferentiated hESCs does not affect the stemness of cells and induces only minor changes to the proliferation rate, cell-cycle profile, and apoptosis. After differentiation is induced, however, the downregulation of those proteins has important effects on proliferation, apoptosis, telomerase activity, and the efficiency of differentiation toward the neuroectodermal lineage. Furthermore, those processes are affected only when one, but not both, of the two proteins is downregulated; the knockdown of both HMGB1 and HMGB2 results in a normal phenotype. Those results advance our knowledge of regulation of hESC and human neuroectodermal cell differentiation and illustrate the distinct roles of HMGB1 and HMGB2 during early human development.
Asunto(s)
Diferenciación Celular , Proteína HMGB1/metabolismo , Proteína HMGB2/metabolismo , Histonas/metabolismo , Células Madre Embrionarias Humanas/citología , Células Madre Embrionarias Humanas/metabolismo , Apoptosis/genética , Ciclo Celular/genética , Línea Celular , Linaje de la Célula/genética , Proliferación Celular/genética , Autorrenovación de las Células/genética , Forma de la Célula/genética , Regulación hacia Abajo/genética , Humanos , Placa Neural/citología , Telomerasa/metabolismo , TransfecciónRESUMEN
HMGB1 protein and linker histone H1 have overlapping binding sites in the nucleosome. HMGB1 has been implicated in many DNA-dependent processes in chromatin involving binding of specific proteins, including transcription factors, to DNA sites pre-bent by HMGB1. HMGB1 can also act as an extracellular signaling molecule by promoting inflammation, tumor growth a metastasis. Many of the intra- and extracellular functions of HMGB1 depend on redox-sensitive cysteine residues of the protein. Here we report that mild oxidization of HMGB1 (and much less mutation of cysteines involved in disulphide bond formation) can severely compromise the functioning of the protein as a DNA chaperone by inhibiting its ability to unwind or bend DNA. Histone H1 (via the highly basic C-terminal domain) significantly inhibits DNA bending by the full-length HMGB1, and the inhibition is further enhanced upon oxidization of HMGB1. Interestingly, DNA bending by HMGB1 lacking the acidic C-tail (HMGB1ΔC) is much less affected by histone H1, but oxidization rendered DNA bending by HMGB1ΔC and HMGB1 equally prone for inhibition by histone H1. Possible consequences of histone H1-mediated inhibition of DNA bending by HMGB1 of different redox state for the functioning of chromatin are discussed.
Asunto(s)
Cisteína/metabolismo , ADN Superhelicoidal/metabolismo , Proteína HMGB1/metabolismo , Histonas/metabolismo , Animales , Cisteína/genética , Proteína HMGB1/química , Proteína HMGB1/genética , Histonas/química , Histonas/genética , Modelos Moleculares , Mutación , Nucleosomas , Oxidación-Reducción , Unión Proteica , RatasRESUMEN
HMGB1 is an architectural protein in chromatin, acting also as a signaling molecule outside the cell. Recent reports from several laboratories provided evidence that a number of both the intracellular and extracellular functions of HMGB1 may depend on redox-sensitive cysteine residues of the protein. In this study we demonstrate that redox state of HMGB1 can significantly modulate the ability of the protein to bind and bend DNA, as well as to promote DNA end-joining. We also report a high affinity binding of histone H1 to hemicatenated DNA loops and DNA minicircles. Finally, we show that reduced HMGB1 can readily displace histone H1 from DNA, while oxidized HMGB1 has limited capacity for H1 displacement. Our results suggested a novel mechanism for the HMGB1-mediated modulation of histone H1 binding to DNA. Possible biological consequences of linker histones H1 replacement by HMGB1 for the functioning of chromatin are discussed.
Asunto(s)
Cromatina/metabolismo , ADN Circular/metabolismo , ADN Concatenado/metabolismo , Proteína HMGB1/metabolismo , Histonas/metabolismo , Proteínas Recombinantes/metabolismo , Animales , Bovinos , Cromatina/genética , ADN Circular/genética , ADN Concatenado/genética , Expresión Génica , Vectores Genéticos/química , Proteína HMGB1/genética , Histonas/genética , Humanos , Oxidación-Reducción , Unión Proteica , Ratas , Proteínas Recombinantes/genéticaRESUMEN
Telomere repeats are added onto chromosome ends by telomerase, consisting of two main core components: a catalytic protein subunit (telomerase reverse trancriptase, TERT), and an RNA subunit (telomerase RNA, TR). Here, we report for the first time evidence that HMGB1 (a chromatin-associated protein in mammals, acting as a DNA chaperone in transcription, replication, recombination, and repair) can modulate cellular activity of mammalian telomerase. Knockout of the HMGB1 gene (HMGB1 KO) in mouse embryonic fibroblasts (MEFs) results in chromosomal abnormalities, enhanced colocalization of γ-H2AX foci at telomeres, and a moderate shortening of telomere lengths. HMGB1 KO MEFs also exhibit significantly (>5-fold) lower telomerase activity than the wild-type MEFs. Correspondingly, enhanced telomerase activity is observed upon overexpression of HMGB1 in MEFs. HMGB1 physically interacts with both TERT and TR, as well as with active telomerase complex in vitro. However, direct interaction of HMGB1 with telomerase is most likely not accountable for the observed higher telomerase activity in HMGB1-containing cells, as revealed from the inability of purified HMGB1 protein to stimulate telomerase activity in vitro. While no transcriptional silencing of TERT is observed in HMGB1 KO MEFs, levels of TR are diminished (~3-fold), providing possible explanation for the observed lower telomerase activity in HMGB1 KO cells. Interestingly, knockout of the HMGB2 gene elevates telomerase activity (~3-fold) in MEFs, suggesting that the two closely related proteins of the HMGB family, HMGB1 and HMGB2, have opposite effects on telomerase activity in the cell. The ability of HMGB1 to modulate cellular activity of telomerase and to maintain telomere integrity can help to understand some aspects of the protein involvement in chromosome stability and cancer.
Asunto(s)
Fibroblastos/citología , Técnicas de Inactivación de Genes , Proteína HMGB1/genética , ARN/genética , Telomerasa/genética , Animales , Línea Celular , Aberraciones Cromosómicas , Daño del ADN , Fragmentación del ADN , Replicación del ADN , Regulación hacia Abajo , Fibroblastos/metabolismo , Proteína HMGB1/metabolismo , Proteína HMGB2/genética , Proteína HMGB2/metabolismo , Histonas/genética , Histonas/metabolismo , Hibridación Fluorescente in Situ , Ratones , Microscopía Fluorescente , ARN/metabolismo , Telomerasa/metabolismo , Telómero/metabolismo , Telómero/patologíaRESUMEN
HMGB proteins are members of the High Mobility Group (HMG) superfamily, possessing a unique DNA-binding domain, the HMG-box, which can bind non-B-type DNA structures (bent, kinked and unwound) with high affinity, and also distort DNA by bending/looping and unwinding. HMGBs (there are four HMGBs in mammals, HMGB1-4) are highly abundant and ubiquitously expressed non-histone proteins, acting as DNA chaperones influencing multiple processes in chromatin such as transcription, replication, recombination, DNA repair and genomic stability. Although HMGB1 is a nuclear protein, it can be secreted into the extracellular milieu as a signaling molecule when cells are under stress, in particular, when necrosis occurs. Mammalian HMGBs contain two HMG-boxes arranged in tandem, share more than 80% identity and differ in the length (HMGB1-3) or absence (HMGB4) of the acidic C-tails. The acidic tails consist of consecutive runs of only Glu/Asp residues of various length, and modulate the DNA-binding properties and functioning of HMGBs. HMGBs are subject to post-translational modifications which can fine-tune interactions of the proteins with DNA/chromatin and determine their relocation from the nucleus to the cytoplasm and secretion. Association of HMGBs with chromatin is highly dynamic, and the proteins affect the chromatin fiber as architectural factors by transient interactions with nucleosomes, displacement of histone H1, and facilitation of nucleosome remodeling and accessibility of the nucleosomal DNA to transcription factors or other sequence-specific proteins.
Asunto(s)
Cromatina/metabolismo , ADN/metabolismo , Proteínas HMGB/metabolismo , Secuencia de Aminoácidos , Animales , Regulación de la Expresión Génica , Proteínas HMGB/química , Proteínas HMGB/genética , Humanos , Datos de Secuencia Molecular , Unión Proteica , Procesamiento Proteico-PostraduccionalRESUMEN
Topoisomerase IIalpha (topo IIalpha) is a nuclear enzyme involved in several critical processes, including chromosome replication, segregation and recombination. Previously we have shown that chromosomal protein HMGB1 interacts with topo IIalpha, and stimulates its catalytic activity. Here we show the effect of HMGB1 on the activity of the human topo IIalpha gene promoter in different cell lines. We demonstrate that HMGB1, but not a mutant of HMGB1 incapable of DNA bending, up-regulates the activity of the topo IIalpha promoter in human cells that lack functional retinoblastoma protein pRb. Transient over-expression of pRb in pRb-negative Saos-2 cells inhibits the ability of HMGB1 to activate the topo IIalpha promoter. The involvement of HMGB1 and its close relative, HMGB2, in modulation of activity of the topo IIalpha gene is further supported by knock-down of HMGB1/2, as evidenced by significantly decreased levels of topo IIalpha mRNA and protein. Our experiments suggest a mechanism of up-regulation of cellular expression of topo IIalpha by HMGB1/2 in pRb-negative cells by modulation of binding of transcription factor NF-Y to the topo IIalpha promoter, and the results are discussed in the framework of previously observed pRb-inactivation, and increased levels of HMGB1/2 and topo IIalpha in tumors.
Asunto(s)
Antígenos de Neoplasias/genética , ADN-Topoisomerasas de Tipo II/genética , Proteínas de Unión al ADN/genética , Proteína HMGB1/metabolismo , Proteína HMGB2/metabolismo , Regulación hacia Arriba , Anciano , Antígenos de Neoplasias/biosíntesis , Factor de Unión a CCAAT/metabolismo , Línea Celular Tumoral , ADN/química , ADN/metabolismo , ADN-Topoisomerasas de Tipo II/biosíntesis , Proteínas de Unión al ADN/biosíntesis , Proteína HMGB1/química , Proteína HMGB1/genética , Humanos , Masculino , Mutagénesis , Regiones Promotoras Genéticas , Proteína de Retinoblastoma/metabolismo , Activación TranscripcionalRESUMEN
DNA topoisomerase IIalpha (topo IIalpha) is an essential nuclear enzyme and its unique decatenation activity has been implicated in many aspects of chromosome dynamics such as chromosome replication and segregation during mitosis. Here we show that chromatin-associated protein HMGB1 (a member of the large family of HMG-box proteins with possible functions in DNA replication, transcription, recombination and DNA repair) promotes topo IIalpha-mediated catenation of circular DNA, relaxation of negatively supercoiled DNA and decatenation of kinetoplast DNA. HMGB1 interacts with topo IIalpha and this interaction, like the stimulation of the catalytic activity of the enzyme, requires both HMG-box domains of HMGB1. A mutant of HMGB1, which cannot change DNA topology stimulates DNA decatenation by topo IIalpha indistinguishably from the wild-type protein. Although HMGB1 stimulates ATP hydrolysis by topo IIalpha, the DNA cleavage is much more enhanced. The observed abilities of HMGB1 to interact with topo IIalpha and promote topo IIalpha binding to DNA suggest a mechanism by which HMGB1 stimulates the catalytic activity of the enzyme via enhancement of DNA cleavage.
Asunto(s)
Antígenos de Neoplasias/metabolismo , ADN-Topoisomerasas de Tipo II/metabolismo , Proteínas de Unión al ADN/metabolismo , ADN/metabolismo , Proteínas del Grupo de Alta Movilidad/metabolismo , Proteínas Represoras/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Catálisis , ADN/química , ADN/ultraestructura , ADN Circular/metabolismo , ADN de Cinetoplasto/metabolismo , ADN Superhelicoidal/metabolismo , Dicetopiperazinas , Electroforesis en Gel de Agar , Inhibidores Enzimáticos/farmacología , Proteína HMGB1 , Humanos , Conformación de Ácido Nucleico , Piperazinas/farmacología , RatasRESUMEN
The parasitic helminth Schistosoma mansoni contains three HMGB proteins, HMGB1, HMGB2 and HMGB3, of primary amino acid sequences highly similar to vertebrate proteins. In this report we describe the characterization of the HMGB1 proteins and their genes from S. mansoni and Schistosoma japonicum. The deduced amino acid sequences of HMGB1 proteins from both schistosome species are identical, and comprise 176 residues. The proteins contain the two evolutionarily highly conserved HMG-box domains, A and B, exhibiting 60% similarity to mammalian HMGB1. Unlike the human HMGB1 which contains an unbroken run of 30 glutamic or aspartic residues, the SmHMGB1 or SjHMGB1 proteins possess unusually short acidic C-terminal tails (5 acidic residues interrupted by 2 serines). Southern hybridization and DNA sequencing revealed a single copy HMGB1 gene, composed of 3 exons and two introns, in S. mansoni. The exon/intron boundaries are identical to those of the human HMGB1 gene, with the exception that the second exon of the SmHMGB1 gene which is not split into two exons as in the human HMGB1 gene. RNA blot analysis revealed that the SmHMGB1 gene is constitutively expressed in similar levels both in male and female worms. The single-sized mRNA for SmHMGB1 is consistent with the size derived from the cDNA. Although DNA binding properties of SmHMGB1 (or SjHMGB1) protein seem to be similar to those previously reported with human HMGB1, i.e., preferential binding to supercoiled DNA over linear DNA, specific recognition of DNA four-way junctions, DNA-induced supercoiling in the presence of topoisomerase I, and DNA bending, we have observed two important differences relative to those observed with the human HMGB1: (i) the inability of the isolated SmHMGB1 domain A to bend DNA (as revealed by T4 ligase-mediated circularization assay), and (ii) higher DNA supercoiling and bending potential of the SmHMGB1 protein as compared to its human counterpart. The latter finding may indicate that the long acidic C-tail of human HMGB1 has much stronger repressive role on DNA bending or DNA supercoiling by topoisomerase I at physiological ionic strength than the short C-tail of the SmHMGB1 protein. Considering the important role of HMGB1 in DNA replication, transcription, recombination, and in particularly, the mediation of inflammation responses in mammalian cells, further studies on schistosome HMGB proteins may provide valuable information related to schistosomiasis, where inflammation plays a critical role in this disease.
Asunto(s)
Proteína HMGB1/genética , Proteína HMGB1/metabolismo , Proteínas del Helminto/genética , Proteínas del Helminto/metabolismo , Schistosoma japonicum/genética , Schistosoma japonicum/metabolismo , Schistosoma mansoni/genética , Schistosoma mansoni/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , ADN Complementario/genética , ADN de Helmintos/química , ADN de Helmintos/genética , ADN de Helmintos/metabolismo , ADN Superhelicoidal/química , ADN Superhelicoidal/genética , ADN Superhelicoidal/metabolismo , Genes de Helminto , Humanos , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Schistosoma japonicum/patogenicidad , Schistosoma mansoni/patogenicidad , Homología de Secuencia de AminoácidoRESUMEN
Protein HMGB1 has long been known as one of the most abundant non-histone proteins in the nucleus of mammalian cells, and has regained interest recently for its function as an extracellular cytokine. As a DNA-binding protein, HMGB1 facilitates DNA-protein interactions by increasing the flexibility of the double helix, and binds specifically to distorted DNA structures. We have previously observed that HMGB1 binds with extremely high affinity to a novel DNA structure, hemicatenated DNA loops (hcDNA), in which double-stranded DNA fragments containing a tract of poly(CA).poly(TG) form a loop maintained at its base by a hemicatenane. Here, we show that the single HMGB1 domains A and B, the HMG-box domain of sex determination factor SRY, as well as the prokaryotic HMGB1-like protein HU, specifically interact with hcDNA (Kd approximately 0.5 nM). However, the affinity of full-length HMGB1 for hcDNA is three orders of magnitude higher (Kd<0.5 pM) and requires the simultaneous presence of both HMG-box domains A and B plus the acidic C-terminal tail on the molecule. Interestingly, the high affinity of the full-length protein for hcDNA does not decrease in the presence of magnesium. Experiments including a comparison of HMGB1 binding to hcDNA and to minicircles containing the CA/TG sequence, binding studies with HMGB1 mutated at intercalating amino acid residues (involved in recognition of distorted DNA structures), and exonuclease III footprinting, strongly suggest that the hemicatenane, not the DNA loop, is the main determinant of the affinity of HMGB1 for hcDNA. Experiments with supercoiled CA/TG-minicircles did not reveal any involvement of left-handed Z-DNA in HMGB1 binding. Our results point to a tight structural fit between HMGB1 and DNA hemicatenanes under physiological conditions, and suggest that one of the nuclear functions of HMGB1 could be linked to the possible presence of hemicatenanes in the cell.
Asunto(s)
ADN Encadenado/química , ADN Encadenado/metabolismo , Proteína HMGB1/química , Proteína HMGB1/metabolismo , Sitios de Unión , Células Cultivadas , Proteínas ELAV/química , Dominios HMG-Box , Células HeLa , Humanos , Conformación de Ácido Nucleico , Unión Proteica , Estructura Terciaria de Proteína , Proteína de la Región Y Determinante del Sexo/químicaRESUMEN
We have recently observed that chromatin architectural protein HMGB1 (previously reported to be involved in numerous biological processes such as DNA replication, recombination, repair, tumor growth, and metastasis) could bind with extremely high affinity (K(d) < 1 pM) to a novel DNA structure that forms a DNA loop maintained at its base by a hemicatenane (hcDNA). The loop of hcDNA contains a track of repetitive sequences derived from CA-microsatellites. Here, we report using a gel-retardation assay that tumor-suppressor protein p53 can also bind to hcDNA. p53 is a crucial molecule protecting cells from malignant transformation by regulating cell-cycle progression, apoptosis, and DNA repair by activation or repression of transcription of its target genes by binding to specific p53 DNA-binding sites and/or certain types of DNA lesions or alternative DNA structures. The affinity of p53 for hcDNA (containing sequences with no resemblance to the p53 DNA consensus sequence) is >40-fold higher (K(d) approximately 0.5 nM) than that for its natural specific binding sites within its target genes (Mdm2 promoter). Binding of p53 to hcDNA remains detectable in the presence of up to approximately 4 orders of magnitude of mass excess of competitor linear DNA, suggesting a high specificity of the interaction. p53 displays a higher affinity for hcDNA than for DNA minicircles (lacking functional p53-specific binding sequence) with a size similar to that of the loop within the hcDNA, indicating that the extreme affinity of p53 for hcDNA is likely due to the binding of the protein to the hemicatenane. Although binding of p53 to hcDNA occurs in the absence of the nonspecific DNA-binding extreme carboxy-terminal regulatory domain (30-C, residues 363-393), the isolated 30-C domain (but not the sequence-specific p53 "core domain", residues 94-312) can also bind hcDNA. Only the full-length p53 can form stable ternary complexes with hcDNA and HMGB1. The possible biological relevance of p53 and HMGB1 binding to hemicatenanes is discussed.
Asunto(s)
ADN Encadenado/química , Proteína HMGB1/química , Regiones Promotoras Genéticas/genética , Proteína p53 Supresora de Tumor/química , Animales , Sitios de Unión , Bovinos , ADN Encadenado/metabolismo , Ensayo de Cambio de Movilidad Electroforética , Glutatión Transferasa/genética , Glutatión Transferasa/metabolismo , Proteína HMGB1/metabolismo , Humanos , Conformación de Ácido Nucleico , Unión Proteica/fisiología , Estructura Terciaria de Proteína/fisiología , Ratas , Especificidad por Sustrato , Timo/citología , Activación Transcripcional , Proteína p53 Supresora de Tumor/metabolismo , Proteínas Supresoras de Tumor/metabolismoRESUMEN
Several proteins that specifically bind to DNA modified by cisplatin, including those containing HMG-domains, mediate antitumor activity of this drug. Oligodeoxyribonucleotide duplexes containing a single, site-specific interstrand cross-link of cisplatin were probed for recognition by the rat chromosomal protein HMGB1 and its domains A and B using the electrophoretic mobility-shift assay. It has been found that the full-length HMGB1 protein and its domain B to which the lysine-rich region (seven amino acid residues) of the A/B linker is attached at the N-terminus (the domain HMGB1b7) specifically recognize DNA interstrand cross-linked by cisplatin. The affinity of these proteins to the interstrand cross-link of cisplatin is not very different from that to the major 1,2-GG intrastrand cross-link of this drug. In contrast, no recognition of the interstrand cross-link by the domain B lacking this region or by the domain A with or without this lysine-rich region attached to its C-terminus is noticed under conditions when these proteins readily bind to 1,2-GG intrastrand adduct. A structural model for the complex formed between the interstrand cross-linked DNA and the domain HMGB1b7 was constructed and refined using molecular mechanics and molecular dynamics techniques. The calculated accessible areas around the deoxyribose protons correlate well with the experimental hydroxyl radical footprint. The model suggests that the only major adaptation necessary for obtaining excellent surface complementarity is extra DNA unwinding (approximately 40 degrees ) at the site of the cross-link. The model structure is consistent with the hypothesis that the enhancement of binding affinity afforded by the basic lysine-rich A/B linker is a consequence of its tight binding to the sugar-phosphate backbone of both DNA strands.
Asunto(s)
Antineoplásicos/química , Cisplatino/química , Reactivos de Enlaces Cruzados/química , Aductos de ADN/química , Proteína HMGB1/química , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Aminoácidos/química , Animales , Emparejamiento Base , Desoxirribosa/química , Proteína HMGB1/aislamiento & purificación , Enlace de Hidrógeno , Radical Hidroxilo/química , Lisina/química , Modelos Moleculares , Datos de Secuencia Molecular , Ácidos Nucleicos Heterodúplex/química , Fragmentos de Péptidos/química , Unión Proteica , Huella de Proteína , Estructura Terciaria de Proteína , Protones , Ratas , Solventes , Propiedades de SuperficieRESUMEN
The recently cloned gene p73 is a close homologue of p53, which is a crucial tumor suppressor gene for preventing the malignant transformation of cells by inducing cell cycle arrest and apoptosis. Previous reports have shown that architectural DNA-bending/looping chromosomal proteins HMGB1 and HMGB2 (formerly known as HMG1 and HMG2), which function in a number of biological processes including transcription and DNA repair, interact in vitro with p53 and stimulate p53 binding to DNA containing p53 consensus sites. Here, we report that HMGB1 physically interacts with two splicing variants of p73, alpha and beta (pull-down assay), and enhances binding of p73 to specific cognate DNA sites (gel-shift assay). Both HMG box domains of HMGB1, A and B, interact with p73alpha. Association of HMGB1 with p73, like the demonstrated ability of HMGB1 to stimulate p73 binding to different p53-responsive elements, requires the oligomerization region and/or region between DNA-binding domain and oligomerization domain of p73 (residues 312-381). Transient transfections revealed that ectopically expressed or endogenous HMGB1 and HMGB2 (antisense strategy) significantly inhibit in vivo both p73alpha/beta- and p53-dependent transactivation from the Bax gene promoter (and much less from Mdm2 and p21(waf1) promoters) in p53-deficient SAOS-2 cells. In contrast, HMGB1 and HGMB2 stimulate p73- or p53-dependent transactivation in p53-deficient H1299 cells, irrespective of the promoter used. Our results suggest that ubiquitously expressed HMGB1 and HMGB2 have potential to cell- and promoter-specifically down- or up-regulate in vivo transcriptional activity of different members of the p53 family. A possible mechanism of HMGB1-mediated modulation of p73- and p53-dependent transactivation is discussed.