RESUMEN
INTRODUCTION: Membranoproliferative glomerulonephritis type II or dense deposit disease (MPGN II/DDD) causes chronic renal dysfunction that progresses to end stage renal disease in about half of patients within 10 years of diagnosis. Deficiency of and mutations in the complement factor H (CFH) gene are associated with the development of MPGN II/DDD, suggesting that dysregulation of the alternative pathway of the complement cascade is important in disease pathophysiology. SUBJECTS: Patients with MPGN II/DDD were studied to determine whether specific allele variants of CFH and CFHR5 segregate preferentially with the MPGN II/DDD disease phenotype. The control group was compromised of 131 people in whom age related macular degeneration had been excluded. RESULTS: Allele frequencies of four single nucleotide polymorphisms in CFH and three in CFHR5 were significantly different between MPGN II/DDD patients and controls. CONCLUSION: We have identified specific allele variants of CFH and CFHR5 associated with the MPGN II/DDD disease phenotype. While our data can be interpreted to further implicate complement in the pathogenesis of MPGN II/DDD, these associations could also be unrelated to disease pathophysiology. Functional studies are required to resolve this question.
Asunto(s)
Proteínas Sanguíneas/genética , Factor H de Complemento/genética , Variación Genética , Glomerulonefritis Membranoproliferativa/genética , Biopsia , Proteínas del Sistema Complemento , Cartilla de ADN , Eliminación de Gen , Frecuencia de los Genes , Glomerulonefritis Membranoproliferativa/clasificación , Glomerulonefritis Membranoproliferativa/patología , Humanos , Mutación , Reacción en Cadena de la Polimerasa , Polimorfismo de Nucleótido Simple , Valores de ReferenciaRESUMEN
In many differentiating cells, a reduction of c-myc proto-oncogene expression is a prerequisite for terminal differentiation. The downmodulation of c-myc in differentiating cells is due to at least two different mechanisms: (i) an elongation block to c-myc transcription activated during an early phase of differentiation and (ii) an inhibition of transcription initiation activated during a later phase. In order to determine cis-acting target structures of the c-myc gene required for the late-phase downregulation of transcriptional initiation, we permanently transfected U937 cells with constructs containing the bacterial chloramphenicol acetyl transferase (CAT) gene driven by a 2.8 kb c-myc promoter region or deletions thereof. We determined two distinct domains in the c-myc promoter region both of which are essential for efficient terminal downregulation: a proximal domain and a distal domain which are located between base pairs -606 to -101, and between -2392 to -1396, respectively, relative to P1. The identification of two distinct regulatory elements suggests the requirement and cooperation of two regulatory factors as an essential event for mediating differentiation-induced downregulation of c-myc in monocytic cells. The implications of these results for deregulation of the translocated c-myc allele in Burkitt's lymphoma are discussed.
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Genes Reguladores , Genes myc , Transcripción Genética , Diferenciación Celular , Cloranfenicol O-Acetiltransferasa/genética , Regulación hacia Abajo , Humanos , Leucemia Monocítica Aguda/genética , Leucemia Monocítica Aguda/patología , Regiones Promotoras Genéticas , Proto-Oncogenes Mas , Células Tumorales CultivadasRESUMEN
The human EGR-4 (AT133) gene represents one member of a family of four related zinc finger proteins, that are simultaneously and coordinately induced in resting cells upon growth stimulation. In order to characterise the function of the EGR-4 zinc finger protein, we have expressed the protein in the eukaryotic baculovirus system. The recombinant EGR-4 protein has a molecular mass of 78 kDa, as demonstrated by SDS-PAGE and Western blotting. DNA binding studies revealed that the EGR-4 protein binds to the EGR consensus motif GCGTGGGCG, but not to the G-rich regulatory ZIP-element of the human IL-2 gene, that is a binding site for EGR-1. EGR-4 functions as transcriptional repressor. Overexpression of EGR-4 mediates repression of a minimal c-fos promoter through a threefold EGR consensus site. Furthermore the EGR-4 protein displays autoregulatory activities. This protein downregulates expression of its own gene promoter in a dose dependent manner. A G-rich region in the EGR-4 promoter, located at position -106 to -82, could be identified as binding site for the recombinant EGR-4 protein. A comparison of the two related zinc finger proteins EGR-4 and EGR-1 revealed for each protein distinct and specific DNA binding- and transcriptional regulatory activities.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica , Proteínas Inmediatas-Precoces , Proteínas Represoras/metabolismo , Transcripción Genética , Dedos de Zinc , Animales , Baculoviridae/genética , Sitios de Unión , Western Blotting , Línea Celular , Clonación Molecular , Secuencia de Consenso , ADN/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Proteína 1 de la Respuesta de Crecimiento Precoz , Factores de Transcripción de la Respuesta de Crecimiento Precoz , Expresión Génica , Genes Reporteros , Humanos , Células Jurkat , Regiones Promotoras Genéticas , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Represoras/química , Proteínas Represoras/genética , Spodoptera , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Dedos de Zinc/genéticaRESUMEN
BACKGROUND: The aetiology of atypical haemolytic uraemic syndrome (aHUS) is, in contrast to classical, Shiga-like toxin induced HUS in children, largely unknown. Deficiency of human complement factor H and familial occurrence led to identification of the factor H gene (FH1) as the susceptibility gene, but the frequency and relevance of FH1 mutations are unknown. METHODS: We established a German registry for aHUS and analysed in all patients and 100 controls the complete FH1 gene by single strand confirmational polymorphism and DNA sequencing. In addition, complement C3 and factor H serum levels were assayed. Demographic data at onset of aHUS and follow up were compared for the mutation positive and negative groups. RESULTS: Of 111 patients with aHUS (68 female, 43 male, mean age 33 years) 14% had FH1 germline mutations, including two of eight patients with familial aHUS. For each of these eight patients, both parents were tested, and we were able to trace the mutation for five cases. In the other three cases (one with the mutation 3749 C/T, one with 3200 T/C, and one with 3566+1 G/A), we could not detect the mutation in either parent, although paternity was proven by genetic fingerprinting, suggesting that these subjects have new mutations. C3 was decreased in five mutation carriers but also in two non-carriers, and factor H was decreased in none of the carriers, but elevated in six carriers and 15 non-carriers. Clinical parameters including associated medications and diseases, and outcome of aHUS and of post-aHUS kidney transplantation were similar in the mutation positive and negative groups. CONCLUSION: FH1 germline mutations occur with considerable frequency in patients with aHUS. Hypocomplementaemia is not regularly associated with a germline mutation, and factor H serum levels can even be elevated. Screening for FH1 mutations contributes to the classification of aHUS.
Asunto(s)
Factor H de Complemento/genética , Síndrome Hemolítico-Urémico/genética , Adulto , Austria , Complemento C3/metabolismo , Factor H de Complemento/metabolismo , ADN/química , ADN/genética , Análisis Mutacional de ADN , Ensayo de Inmunoadsorción Enzimática , Femenino , Alemania , Síndrome Hemolítico-Urémico/sangre , Síndrome Hemolítico-Urémico/complicaciones , Humanos , Italia , Fallo Renal Crónico/complicaciones , Fallo Renal Crónico/terapia , Trasplante de Riñón , Masculino , Mutación , Polimorfismo Conformacional Retorcido-Simple , Sistema de Registros/estadística & datos numéricos , SuizaRESUMEN
The Early Growth Response Genes (EGR-1 to AT133/EGR-4) encode a family of proteins that are composed of three homologous consecutive zinc fingers of the Cys2-His2 type and different flanking sequence. Upon growth stimulation of resting cells the four EGR-genes are simultaneously transcribed. We have analyzed the expression of the four EGR-proteins in Jurkat T cells and show by Western blot analysis that the four EGR-proteins are coordinately induced upon treatment with a combination of PHA and PMA. As the individual proteins are reported to bind to identical target sequences, we have analyzed the DNA-binding of the native proteins. Using nuclear extract in which we have demonstrated expression of all four EGR-proteins, only EGR-1, but no other member of this protein family is found to bind to the EGR-consensus site (GCG GGG GCG). In addition, DNA-binding of both native EGR-1 and of recombinant EGR-1 and AT133/EGR-4 proteins expressed in insect cells was analyzed. This comparison revealed distinct binding properties of recombinant EGR-1 and AT133/EGR-4 to oligonucleotides that include the EGR-consensus sites. The distinct binding affinities suggest that in vivo EGR-proteins bind to different target sequences and that each EGR-protein regulates distinct target genes. This is underlined by demonstrating that EGR-1 but not AT133/EGR-4 binds to a related G-rich promoter element with the sequence GGG GTG GGG. This G-rich sequence serves as an overlapping binding site for the two zinc finger proteins EGR-1 and Sp1. As similar overlapping binding sites for EGR-1 and Sp1 have been identified in several human and mouse gene promoters, we raise the question whether the Sp1 binding sites described in a large number of eukaryotic gene promoters also represent binding sites for EGR-1.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteínas Inmediatas-Precoces , Oligodesoxirribonucleótidos/metabolismo , Factores de Transcripción/metabolismo , Dedos de Zinc , Animales , Sitios de Unión , Extractos Celulares , Línea Celular , Secuencia de Consenso , Proteínas de Unión al ADN/biosíntesis , Proteínas de Unión al ADN/genética , Proteína 1 de la Respuesta de Crecimiento Precoz , Proteína 2 de la Respuesta de Crecimiento Precoz , Proteína 3 de la Respuesta de Crecimiento Precoz , Factores de Transcripción de la Respuesta de Crecimiento Precoz , Genes Sobrepuestos , Humanos , Interleucina-2/genética , Células Jurkat , Mitógenos/farmacología , Fitohemaglutininas/farmacología , Regiones Promotoras Genéticas , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Factor de Transcripción Sp1/metabolismo , Spodoptera/citología , Acetato de Tetradecanoilforbol/farmacología , Factores de Transcripción/biosíntesis , Factores de Transcripción/genéticaRESUMEN
Borrelia burgdorferi, the causative agent of Lyme disease, differ in their susceptibility to normal human serum and are consequently classified as complement-resistant, complement-sensitive and intermediate complement-sensitive. Most isolates belonging to the genospecies B. afzelii are complement-resistant, while particularly B. garinii isolates were rapidly killed by complement. In general, isolates of the genospecies B. burgdorferi sensu stricto (s.s.) are intermediate complement-sensitive. Independent of the genospecies, all Borreliae were capable to activate the classical and/or the alternative pathway. Deposition of the activation products C3, C6, and TCC is much stronger by B. burgdorferi s.s. and B. garinii isolates than by B. afzelii isolates. The mechanism(s) on how Borreliae evade complement-mediated bacteriolysis has recently been described by showing that complement-resistant B. afzelii isolates but not the complement-sensitive B. garinii isolates absorb human complement regulators FHL-1/reconectin and factor H. Surface-attached FHL-1/reconectin maintains its complement regulatory activity and supports factor I-mediated C3b cleavage to iC3b. In complement-resistant Borreliae, two outer surface proteins, the 27.5 kDa (CRASP-1, complement regulator-acquiring surface protein 1) and the 20/21 kDa (CRASP-2), are responsible for the surface attachment of the two complement regulators. CRASP-1, which is present in complement-resistant Borreliae, binds preferentially FHL-1/reconectin while CRASP-2, which is restrictively expressed, binds preferentially factor H. Thus, complement-resistant Borreliae bind human complement regulators and control complement activation on their surface and prevent the formation of toxic activation products.
Asunto(s)
Grupo Borrelia Burgdorferi/inmunología , Grupo Borrelia Burgdorferi/patogenicidad , Proteínas del Sistema Complemento/inmunología , Proteínas de la Membrana Bacteriana Externa/inmunología , Vía Alternativa del Complemento , Vía Clásica del Complemento , Humanos , Técnicas In Vitro , Enfermedad de Lyme/inmunología , Enfermedad de Lyme/microbiologíaRESUMEN
Factor H is a 150 kDa single chain plasma glycoprotein that plays a pivotal role in the regulation of the alternative pathway of complement. Primary sequence analysis reveals a structural organization of this plasma protein, in 20 homologous units, called Short Consensus Repeats (SCRs), each about 60 amino acids long. Biochemical and genetic studies show an association between factor H deficiency and human diseases, including Systemic Lupus Erythematosus, susceptibility to pyogenic infection and a form of membranoproliferative glomerulonephropathy. More recently, factor H deficiency has also been associated with susceptibility to Hemolytic Uremic Syndrome (HUS), a disease consisting of microangiopathic hemolytic anemia, thrombocytopenia and acute renal failure, caused by platelet thrombi which mainly, but not exclusively, form in the microcirculation of the kidney. In this review, we summarize recent genetic and biochemical data, which indicate a critical role for factor H in the pathogenesis of HUS and suggest an important role of the most C-terminal domain, i.e. SCR 20, in the disease. In addition, we discuss the physiological consequences of these findings, as novel functional data show a particular essential role of SCR 20 of factor H as the central discriminatory and regulatory site of this multidomain, multifunctional plasma protein.
Asunto(s)
Factor H de Complemento/deficiencia , Síndrome Hemolítico-Urémico/etiología , Síndrome Hemolítico-Urémico/inmunología , Factor H de Complemento/química , Factor H de Complemento/genética , Proteínas del Sistema Complemento/metabolismo , Síndrome Hemolítico-Urémico/genética , Humanos , Mutación , Estructura Terciaria de ProteínaRESUMEN
We report a novel pathomechanism for membranoproliferative glomerulonephritis type II (MPGN II) caused by a mutant Factor H protein expressed in the plasma. Genetic analyses of two patients revealed deletion of a single Lys residue (K224) located within the complement regulatory region in domain 4 of Factor H. This deletion resulted in defective complement control: mutant protein purified from the plasma of patients showed severely reduced cofactor and decay-accelerating activity, as well as reduced binding to the central complement component C3b. However, cell-binding activity of the mutant protein was normal and comparable to wild-type Factor H. The patients are daughters of consanguineous parents. As both patients but also their healthy mother were positive for C3 nephritic factor, the mutant Factor H protein is considered relevant for unrestricted activation of the disease-causing activation of the alternative complement pathway. Replacement of functional Factor H by fresh frozen plasma (10-15 ml/kg/14 days) was well tolerated, prevented so far disease progression in both patients, and is in the long run expected to preserve kidney function.
Asunto(s)
Factor H de Complemento/genética , Factor H de Complemento/metabolismo , Vía Alternativa del Complemento , Glomerulonefritis Membranoproliferativa/genética , Glomerulonefritis Membranoproliferativa/inmunología , Secuencia de Aminoácidos , Niño , Factor Nefrítico del Complemento 3/análisis , Factor Nefrítico del Complemento 3/metabolismo , Factor H de Complemento/análisis , Consanguinidad , Femenino , Humanos , Lisina/química , Lisina/genética , Datos de Secuencia Molecular , Linaje , Plasma/química , Plasma/metabolismo , Estructura Terciaria de Proteína/genética , Eliminación de SecuenciaRESUMEN
The complement inhibitor Factor H has three distinct binding sites for C3b and for heparin, but in solution uses specifically the most C-terminal domain, i.e. short consensus repeats (SCR) 20 for ligand interaction. Two novel monoclonal antibodies (mABs C14 and C18) that bind to the most C-terminal domain SCR 20 completely blocked interaction of Factor H with the ligands C3b, C3d, heparin and binding to endothelial cells. In contrast, several mAbs that bind to the N-terminus and to the middle regions of the molecule showed no or minor inhibitory effects when assayed by enzyme-linked immunosorbent assay (ELISA) and ligand interaction assays. This paradox between a single functional binding site identified for native Factor H versus multiple interaction sites reported for deletion constructs is explained by a compact conformation of the fluid phase protein with one accessible binding site. On zymosan particles mAbs C14 and C18 blocked alternative pathway activation completely. Thus demonstrating that native Factor H makes the first and initial contact with the C terminus, which is followed by N terminally mediated complement regulation. These results are explained by a conformational hypothetical model: the native Factor H protein has a compact structure and only one binding site accessible. Upon the first contact the protein unfolds and exposes the additional binding sites. This model does explain how Factor H mediates recognition functions during complement control and the clustering of disease associated mutations in patients with haemolytic uraemic syndrome that have been reported in the C-terminal recognition domain of Factor H.
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Anticuerpos Monoclonales/inmunología , Sitios de Unión de Anticuerpos/inmunología , Complemento C3b/inmunología , Complemento C3d/inmunología , Factor H de Complemento/genética , Factor H de Complemento/inmunología , Vía Alternativa del Complemento/inmunología , Células Endoteliales/inmunología , Epítopos/inmunología , Heparina/inmunología , Humanos , Ligandos , Modelos Biológicos , Mutación , Conformación Proteica , Zimosan/inmunologíaRESUMEN
Recently, several factor-H-related molecules have been identified in humans and mice, initiating new interest in this group of serum proteins. Although no complement-regulatory activity has been demonstrated yet for the human factor-H-related proteins, the immunological and structural similarities to factor H suggest overlapping functions. Here, Peter Zipfel and Christine Skerka review these similarities and suggest a new nomenclature to identify members of the factor-H-related family.
Asunto(s)
Proteínas Sanguíneas/química , Proteínas Sanguíneas/fisiología , Factor H de Complemento/química , Factor H de Complemento/fisiología , Secuencia de Aminoácidos , Animales , Humanos , Datos de Secuencia Molecular , Homología de Secuencia de AminoácidoRESUMEN
A novel regulator of the alternative complement pathway with functions similar to that of factor H has been identified in human plasma. The cDNA encoding this factor H-like protein 1 (FHL-1/reconectin) was isolated several years ago. Here, Peter Zipfel and Christine Skerka describe functional analyses revealing that this novel complement regulatory protein forms a molecular link between immune defense and cell adhesion.
Asunto(s)
Adhesión Celular , Factor H de Complemento/fisiología , Vía Alternativa del Complemento , Animales , Dominio Catalítico/fisiología , Cromosomas Humanos Par 1/genética , Factor H de Complemento/genética , Humanos , Streptococcus pyogenes/metabolismo , Streptococcus pyogenes/patogenicidadRESUMEN
Human members of a family of structurally related cytokines, which play a role as effectors of inflammation, were analyzed for their expression and regulation in T lymphocytes. Members of this gene family include Platelet Basic Protein (PBP); Platelet Factor 4 (PF-4); IL-8/NAP-1; IP-10, a gamma interferon induced protein; GRO; pAT 464 and pAT 744. In resting T lymphocytes the RNAs of the individual genes could not be detected, but all genes were induced upon stimulation with PHA or with PHA/PMA. The induction of five genes was blocked by the immunosuppresive drug cyclosporin A (CSA), which appears to affect initial events in T cell activation. This expression in T lymphocytes, especially the sensitivity to CSA, indicates a common immunmodulatory role of these structural related proteins.
Asunto(s)
Ciclosporina/farmacología , Interleucina-8/genética , Familia de Multigenes , Linfocitos T/inmunología , Northern Blotting , Línea Celular , Células Cultivadas , Sondas de ADN , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Cinética , Activación de Linfocitos , Familia de Multigenes/efectos de los fármacos , ARN/genética , ARN/aislamiento & purificación , ARN Mensajero/genética , Linfocitos T/efectos de los fármacos , Transcripción Genética/efectos de los fármacosRESUMEN
Two cDNA clones termed H36-1 and H36-2 were isolated from a human liver cDNA library. Clone H36-1 appears to represent the recently isolated human serum proteins h37 and h42, which are two differently glycosylated forms of a protein antigenically related to human complement factor H. The H36-1 deduced protein sequence is 327 amino acid long and possesses a leader sequence. The secreted part of the protein is comprised of five tandem repeating units, termed short consensus repeats (SCRs). SCR 1 and 2 display high homology to the corresponding region of the recently isolated murine factor H-related cDNA clone 13G1. In contrast, the 3'-end of the H36-1 clone shows sequence homology to the 3'-end of human complement factor H. The second clone, H36-2, is nearly identical to H36-1. Within 1148 base pairs, where the two clones overlap, their nucleotide sequences differed at nine positions. One nucleotide exchange in the sequence of H36-2 which was located within SCR 1 creats a stop codon (TAA). Consequently, the corresponding mRNA cannot code for a functional protein, suggesting that this clone is a transcribed pseudogene. These two clones represent new human members of the family of proteins structurally related to complement factor H.
Asunto(s)
Proteínas Sanguíneas/genética , Proteínas Inactivadoras del Complemento C3b/genética , Secuencia de Aminoácidos , Secuencia de Bases , Northern Blotting , Southern Blotting , Clonación Molecular , Factor H de Complemento , ADN/genética , Glicosilación , Humanos , Hígado/química , Datos de Secuencia Molecular , Hibridación de Ácido Nucleico , Sondas de Oligonucleótidos , ARN/análisis , Secuencias Repetitivas de Ácidos Nucleicos , Alineación de Secuencia , Homología de Secuencia de Ácido NucleicoRESUMEN
The human factor H-like protein 1 (FHL-1) is composed of seven repetitive domains (short consensus repeats; SCRs) that are identical in sequence to the seven NH2-terminal SCRs of the complement regulatory protein factor H. We have identified the native FHL-1 protein as a 42-kDa human plasma protein by immunoblotting and by comparing the mobility to that of a recombinant FHL-1 protein. Here, we demonstrate the existence of two distinct co-migrating human plasma proteins that represent the 42-kDa FHL-1 protein and the previously identified 43-kDa factor H-related 1 beta protein. Similar to factor H, the recombinant FHL-1 protein displays cofactor activity in factor I-mediated cleavage of C3b. To identify relevant SCRs of factor H and FHL-1, we recombinantly expressed the domains shared between the two proteins in the baculovirus expression system. Recombinant FHL-1 and all truncated forms that include SCRs 1 to 4 displayed cofactor activity. All four NH2-terminal SCRs are essential, as deletion mutants composed of SCR 1 and 4 only; of SCRs 1, 2, and 4 only, or of SCRs 1, 3, and 4 only were functionally inactive. Similarly, the distance between these individually folding domains is critical for function, as a recombinant protein that had two and four amino acids inserted between SCRs 1 and 2 or between SCRs 3 and 4, respectively, had no activity. These results demonstrate that all four NH2-terminal SCRs of FHL-1 (and of factor H) are required for cofactor activity in factor I-mediated cleavage of C3b, and that the distance between these SCRs is essential.
Asunto(s)
Factor H de Complemento/genética , Proteínas Inactivadoras de Complemento/análisis , Secuencia de Consenso/genética , Receptores de Complemento/genética , Secuencia de Bases , Factor H de Complemento/aislamiento & purificación , Humanos , Datos de Secuencia Molecular , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genéticaRESUMEN
The three genospecies Borrelia burgdorferi, Borrelia garinii, and Borrelia afzelii, all causative agents of Lyme disease, differ in their susceptibilities to human complement-mediated lysis. We recently reported that serum resistance of borrelias correlates largely with their ability to bind the human complement regulators FHL-1/reconectin and factor H. To date, two complement regulator-acquiring-proteins (CRASP-1 and CRASP-2) have been identified in serum-resistant B. afzelii isolates (P. Kraiczy, C. Skerka, M. Kirschfink, V. Brade, and P. F. Zipfel, Eur. J. Immunol. 31:1674-1684, 2001). Here, we present a comprehensive study of the CRASPs detectable in both serum-resistant and intermediate serum-sensitive B. afzelii and B. burgdorferi isolates. These CRASPs were designated according to the genospecies either as BaCRASPs, when derived from B. afzelii, or as BbCRASPs, for proteins identified in B. burgdorferi isolates. Each borrelial isolate expresses distinct CRASPs that can be differentiated by their mobility and binding phenotypes. A detailed comparison reveals overlapping and even identical binding profiles for BaCRASP-1 (27.5 kDa), BbCRASP-1 (25.9 kDa), and BbCRASP-2 (23.2 kDa), which bind FHL-1/reconectin strongly and interact weakly with factor H. In contrast, two B. afzelii proteins (BaCRASP-4 [19.2 kDa] and BaCRASP-5 [22.5 kDa]) and three B. burgdorferi proteins (BbCRASP-3 [19.8 kDa], BbCRASP-4 [18.5 kDa], and BbCRASP-5 [17.7 kDa]) bind factor H but not FHL-1/reconectin. Most CRASPs bind both human immune regulators at their C-terminal ends. Temperature-dependent up-regulation of CRASPs (BaCRASP-1, BaCRASP-2, and BaCRASP-5) is detected in low-passage borrelias cultured at 33 or 37 degrees C compared with those cultured at 20 degrees C. The characterization of the individual CRASPs on the molecular level is expected to identify new virulence factors and potential vaccine candidates.
Asunto(s)
Proteínas Bacterianas/metabolismo , Grupo Borrelia Burgdorferi/inmunología , Proteínas del Sistema Complemento/metabolismo , Proteínas de la Membrana/metabolismo , Sitios de Unión , Proteínas Sanguíneas/metabolismo , Borrelia burgdorferi/inmunología , Proteínas Inactivadoras del Complemento C3b , Factor H de Complemento/metabolismo , Regulación Bacteriana de la Expresión Génica , Humanos , Inmunidad Innata , Enfermedad de Lyme/etiología , Unión Proteica , TemperaturaRESUMEN
Analysis of base composition has proven important for functional gene analysis. By comparing base composition and codon usage between two specific human gene families we were able to show a highly conserved nucleotide distribution among the members of one gene family and a significant difference between the two families. The two groups selected for analysis were the human factor H gene family, which represents six secreted human plasma proteins with functions in immune defense, and a class of four human zinc finger proteins, termed early growth response (EGR) proteins, which represent DNA-binding transcription factors. The nucleotide distribution of each gene family is distinct: members of the factor H gene family represent AT-rich genes, displaying an overall AT nucleotide content of 62.8% and a particular preference for A nucleotides (33.9%). In contrast, the EGR genes are GC-rich (55.9%) and C nucleotides are used in 31.2%. This nucleotide difference affects codon usage among synonymous codons and is considered of biological significance, as it affects DNA stability. The codon preference is particularly high at codon position 3, where each family selects for codons which have the preferred nucleotide at this silent third position. At position 3, A nucleotides are preferred by factor H genes in 36.3% of the 2, 503 codons analyzed, compared to 10% of the 1,876 codons analyzed for the EGR family. In contrast, C nucleotides are used by the EGR family in 48.1%, compared to 16% of the triplets used by the factor H gene family. This comparison of two human gene families shows that nucleotide distribution and codon usage is not uniform within the human organism and the described differences most likely represent selection constraints between the polymorphic factor H and highly conserved EGR genes.
Asunto(s)
Factor H de Complemento/genética , Proteínas de Unión al ADN/genética , Familia de Multigenes , Factores de Transcripción/genética , Composición de Base , Secuencia de Bases , Proteínas Sanguíneas/genética , Codón/genética , ADN/química , ADN/genética , Humanos , ARN Mensajero/genética , Dedos de Zinc/genéticaRESUMEN
Activation of the interleukin 2 (IL-2) gene after antigen recognition is a critical event for T cell proliferation and effector function. Prior studies have identified several transcription factors that contribute to the activity of the IL-2 promoter in stimulated T lymphocytes. Here we describe a novel regulatory element within the IL-2 promoter located immediately upstream of the nuclear factor of activated T cell (NFAT) domain. This region (termed the zinc finger protein binding region (ZIP)) serves as binding site for two differently regulated zinc finger proteins: the constitutively expressed transcription factor Sp1 and the inducible early growth response protein EGR-1. In unstimulated cells which do not secrete IL-2, only Sp1 binds to this region, while in stimulated IL-2 secreting cells the inducible EGR-1 protein recognizes this element. In Jurkat T cells, the ZIP site serves as an activator for IL-2 gene expression, and a combination of ZIP and NFAT binding sites is required for maximal IL-2 promoter activity. These results suggest a critical role of the ZIP site for IL-2 promoter activity.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteínas Inmediatas-Precoces , Interleucina-2/genética , Regiones Promotoras Genéticas , Factor de Transcripción AP-1/metabolismo , Factores de Transcripción/metabolismo , Animales , Secuencia de Bases , Sitios de Unión , Proteína 1 de la Respuesta de Crecimiento Precoz , Regulación de la Expresión Génica , Humanos , Técnicas In Vitro , Ratones , Datos de Secuencia Molecular , Proteínas Nucleares/metabolismo , ARN Mensajero/genética , Proteínas Recombinantes , Alineación de Secuencia , Homología de Secuencia de Ácido Nucleico , Linfocitos T , Transcripción Genética , Células Tumorales Cultivadas , Dedos de ZincRESUMEN
Factor-H-related proteins and the complement regulatory protein factor H represent a family of structurally and immunologically related plasma proteins. The function of the various factor-H-related proteins are currently unclear and under investigation. The newest member of this group of proteins, the factor-H-related protein 4 (FHR-4) has recently been identified as an amphipathic protein, that is present in free form in human plasma and also as a constituent of triglyceride-rich lipoproteins. In plasma FHR-4 occurs exclusively in a dimeric form, that most likely represents a homodimer consisting of two identical FHR-4 monomers. In order to identify the function of the FHR-4 protein we have recombinantly expressed the protein in the baculovirus system. The recombinant protein is detected in the supernatant of infected insect cells, both in its monomeric and dimeric form. Both the native form (86 kDa) and the recombinant (84 kDa) proteins are posttranslationally modified. Lectin staining showed that the differences in the apparent molecular masses are due to distinct types of attached N-carbohydrate side chains. Functional analyses show dose-dependent binding of recombinant FHR-4 to C3b, thus demonstrating a functional relatedness between FHR-4, factor H and FHL-1 and other complement regulators of the RCA gene cluster.
Asunto(s)
Apolipoproteínas/genética , Baculoviridae/genética , Factor H de Complemento/inmunología , Procesamiento Proteico-Postraduccional , Animales , Apolipoproteínas/fisiología , Unión Competitiva , Células Cultivadas , Complemento C3b/inmunología , Complemento C3b/metabolismo , Factor H de Complemento/genética , Dimerización , Glicosilación , Humanos , Lectinas/química , Peso Molecular , Proteínas Recombinantes/genética , Spodoptera , Coloración y Etiquetado , TransfecciónRESUMEN
The early growth response-1 gene (EGR-1) is induced by a wide range of stimuli in diverse cell types; however, EGR-1-regulated genes display a highly restricted pattern of expression. Recently, an overlapping Sp1.EGR-1 binding site has been identified within the interleukin-2 (IL-2) gene promoter directly upstream of the binding site for the nuclear factor of activated T cells (NFAT). We used transfection assays to study how the abundantly and constitutively expressed Sp1 protein and the immediate early EGR-1 zinc finger protein regulate IL-2 gene expression. Here, we identify EGR-1 as an important activator of the IL-2 gene. In Jurkat T cells, EGR-1 but not Sp1 acts as a potent coactivator for IL-2 transcription, and in combination with NFATc, EGR-1 increases transcription of an IL-2 reporter construct 200-fold. Electrophoretic mobility shift assays reveal that recombinant EGR-1 and NFATc bind independently to their target sites within the IL-2 promoter, and the presence of both sites on the same DNA molecule is required for EGR-1.NFATc.DNA complex formation. The transcriptional synergy observed here for EGR-1 and NFATc explains how the abundant nuclear factor EGR-1 contributes to the expression of restrictively expressed genes.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/fisiología , Regulación de la Expresión Génica/fisiología , Proteínas Inmediatas-Precoces , Interleucina-2/genética , Proteínas Nucleares , Linfocitos T/metabolismo , Factores de Transcripción/metabolismo , Factores de Transcripción/fisiología , Transcripción Genética/fisiología , Línea Celular , Proteína 1 de la Respuesta de Crecimiento Precoz , Humanos , Células Jurkat , Factores de Transcripción NFATC , Regiones Promotoras Genéticas , Unión Proteica , Proteínas Recombinantes/genética , Factor de Transcripción Sp1/metabolismoRESUMEN
The expression and regulation of seven human members of a family of related cytokines, which play a role as effectors of inflammation, were analysed in hemopoietic cells and in fibroblasts. In T lymphocytes all genes: platelet basic protein (PBP); platelet factor 4 (PF-4); IL-8/NAP-1; IP-10; GRO; pAT464 and pAT744 were induced by stimulation with phytohemagglutinin and phorbol 12-myristate 13-acetate (PHA/PMA). In contrast to T cells, only some of the genes were induced upon terminal differentiation of pro-monocytic cells and upon serum stimulation of resting fibroblasts. This distinct expression indicates functional differences of the individual proteins. The expression of inflammatory mediators in fibroblasts suggests the involvement of these cells in inflammatory reactions.