RESUMEN
The yeast Vps4 protein (Vps4p) is a member of the AAA protein family (ATPases associated with diverse cellular activities) and a key player in the transport of proteins out of a prevacuolar endosomal compartment. In human cells, we identified two non-allelic orthologous proteins (VPS4-A and VPS4-B) of yeast Vps4p. The human VPS4-A and VPS4-B proteins display a high degree of sequence identity to each other (80 %) and to the yeast Vps4 protein (59 and 60 %, respectively). Yeast cells lacking a functional VPS4 gene exhibit a temperature-sensitive growth defect and mislocalise a carboxypeptidase Y-invertase fusion protein to the cell surface. Heterologous expression of human VPS4 genes in vps4 mutant yeast strains led, in the case of human VPS4-A, to a partial and, in the case of human VPS4-B, to a complete suppression of the temperature-sensitive growth defect. The vacuolar protein sorting defect of vps4 mutant yeast cells was complemented completely by heterologous expressed human VPS4-B protein, and partially by the human VPS4-A protein. Expression of mutant human VPS4-A (E228Q) and VPS4-B (E235Q) proteins, harbouring single amino acid exchanges in their AAA domains, induced dominant-negative vacuolar protein sorting defects in wild-type yeast cells in both cases. Two-hybrid experiments suggest that the human VPS4-A and VPS4-B proteins can form heteromeric complexes, and subcellular localisation experiments indicate that both human VPS4 proteins associate with endosomal compartments in yeast. Based on these results, we conclude that both human VPS4 proteins are involved in intracellular protein trafficking, presumably at a late endosomal protein transport step, similar to the Vps4p in yeast.
Asunto(s)
Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/metabolismo , Secuencia Conservada/genética , Proteínas Represoras , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , ATPasas Asociadas con Actividades Celulares Diversas , Adenosina Trifosfatasas/química , Secuencia de Aminoácidos , Carboxipeptidasas/metabolismo , Catepsina A , Línea Celular , Clonación Molecular , Complejos de Clasificación Endosomal Requeridos para el Transporte , Endosomas/química , Endosomas/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Genes Dominantes , Prueba de Complementación Genética , Humanos , Datos de Secuencia Molecular , Mutación , Fenotipo , Unión Proteica , Transporte de Proteínas , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Alineación de Secuencia , Temperatura , Técnicas del Sistema de Dos Híbridos , Proteínas de Transporte VesicularRESUMEN
Hurpin (protease inhibitor 13; PI13) is the most recently identified member of the ovalbumin family of serine protease inhibitors (serpins). It is expressed in human epidermal keratinocytes and is downregulated by exposure to ultraviolet irradiation. A role for hurpin in the proliferation or differentiation of keratinocytes has been proposed because of its strong expression in proliferating cells and its deregulated expression in the lesional epidermis of psoriatic patients. Here, we report the cloning, chromosomal localization, and complete sequence of the human hurpin gene. By PCR-based screening of the GeneBridge 4 radiation hybrid panel, we mapped the gene to chromosome 18q21.3, close to a known cluster of ov-serpin genes. Using the full-length cDNA for hurpin, we identified two clones from an arrayed genomic P1 placental library that contain the entire hurpin gene. Sequencing revealed that the gene covers 12.253 kb and is comprised of eight exons and seven introns. The exon--intron boundaries are identical in position and phasing to those in other members of the 18q serpin gene cluster, and analysis of hurpin variants indicated that modified functional inhibitors, differing only in the CD interhelical loop, can be generated by differential splicing of exon 3. These data show that hurpin is a typical member of the 18q ovalbumin-serpins most closely related to the serpins squamous-cell carcinoma antigens 1 and 2.
Asunto(s)
Empalme Alternativo , Cromosomas Humanos Par 18 , Psoriasis/metabolismo , Serpinas/genética , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , Exones , Humanos , Intrones , Datos de Secuencia Molecular , Inhibidor 2 de Activador Plasminogénico/genética , ARN Mensajero , Serpinas/metabolismo , Transcripción Genética , Regulación hacia ArribaRESUMEN
Functional as well as structural reorganization takes place in the surrounding and remote brain areas after focal ischemic lesions. In particular, reactive or regenerative processes have been described to occur in the contralateral hemisphere. We used mRNA differential display to gain more insight into the molecular mechanisms underlying this type of neuronal plasticity. Circumscribed unilateral infarcts consistently affecting the forelimb area of the primary motor cortex were induced photochemically in adult male Wistar rats. The lesion produced significant behavioral asymmetry with subsequent partial recovery within 1 week. Cloning the genes with altered expression profiles identified the 20S proteasome subunit C2 as a gene whose expression level is decreased in contralateral homotopic cortex. Semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) revealed approximately twofold lower proteasome C2 mRNA levels in the lesion group as compared with the sham-operated group. The proteasome serves as the central enzyme of non-lysosomal protein degradation. It is responsible for intracellular protein turnover and is critically involved in a variety of regulation processes, such as cell cycle, metabolism and differentiation. Our results suggest that proteasome activity may play also a role in contralateral cortical plasticity occurring after focal cerebral ischemia.
Asunto(s)
Isquemia Encefálica/metabolismo , Infarto Cerebral/metabolismo , Cisteína Endopeptidasas/metabolismo , Lateralidad Funcional/fisiología , Complejos Multienzimáticos/metabolismo , Animales , Conducta Animal , Cisteína Endopeptidasas/genética , Cartilla de ADN , Miembro Anterior/fisiología , Regulación Enzimológica de la Expresión Génica/fisiología , Masculino , Corteza Motora/enzimología , Movimiento/fisiología , Complejos Multienzimáticos/genética , Plasticidad Neuronal/fisiología , Complejo de la Endopetidasa Proteasomal , ARN Mensajero/análisis , Ratas , Ratas WistarRESUMEN
OBJECTIVE AND DESIGN: The effects of the anticytokine interleukin 10 (IL-10) are mediated by specific receptors. In this study we examined the role of the IL-10 receptor (IL-10R) in the pathophysiology of atopic eczema. MATERIALS AND METHODS: For this purpose we analyzed the expression of IL-10R in the skin of patients with acute and chronic atopic eczema in comparison to the expression in healthy individuals using in situ binding experiments with fluorescently labeled IL-10 and semiquantitative reverse transcriptase-PCR specific for IL-10R1. In addition, we studied the influence of the Th2-associated cytokine interleukin-4 (IL-4), the Th1-associated gamma-interferon (IFN-gamma), the immunosuppressive drug FK506, the H1-antagonist loratadine and UVA irradiation on the expression of IL-10R1 in cultured normal human keratinocytes. RESULTS: We found that IL-10 receptor mRNA and protein are strongly downregulated in acute phase atopic lesions. Furthermore we could show that IL-4, IFN-gamma, FK506, loratadine and UVA enhance the mRNA levels of the IL-10R1 in vitro in normal cultured keratinocytes. We could also demonstrate restored IL-10R1 mRNA levels in lesional atopic skin of a patient after UVA1 therapy. CONCLUSIONS: Our results demonstrate for the first time that IL-10 receptors may have a role in the pathogenesis of atopic eczema and its upregulation by FK506 and UVA could explain the therapeutic efficacy of these agents.
Asunto(s)
Dermatitis Atópica/genética , Dermatitis Atópica/fisiopatología , Queratinocitos/efectos de los fármacos , Receptores de Interleucina/genética , Enfermedad Aguda , Adulto , Anciano , Células Cultivadas , Enfermedad Crónica , Citocinas/genética , Citocinas/farmacología , Dermatitis Atópica/radioterapia , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/efectos de la radiación , Humanos , Inmunosupresores/farmacología , Queratinocitos/efectos de la radiación , Persona de Mediana Edad , ARN Mensajero/efectos de los fármacos , ARN Mensajero/metabolismo , ARN Mensajero/efectos de la radiación , Receptores de Interleucina/análisis , Receptores de Interleucina/efectos de los fármacos , Receptores de Interleucina/efectos de la radiación , Receptores de Interleucina-10 , Tacrolimus/farmacología , Terapia UltravioletaRESUMEN
Epidermal keratinocytes are the primary target of the midrange ultraviolet part (UVB, 280-320 nm) of terrestrial sunlight. Analysis of the resulting UV response at the transcriptional level by differential display PCR identified a formerly unrecognized large group of repressed genes. Among those UV-repressible genes, a novel serine proteinase inhibitor (serpin) termed hurpin (HaCaT UV-repressible serpin) has been identified. The isolated full-length cDNAs harbour a 1176 bp open reading frame encoding a potential protein with 391 amino acid residues and a predicted molecular mass of approximately 44 kDa. The novel serpin has nearly 59 % amino acid identity with the squamous cell carcinoma antigen 1 (SCCA1) and squamous cell carcinoma antigen 2 (SCCA2). In addition, it displays all of the structural features unique to the ovalbumin family of serpins (ov-serpins). The amino acid sequence of the hinge region in the reactive site loop suggests that hurpin has the potential for protease inhibition. The putative reactive center P1-P1'residues were identified as Thr356-Ser357 by alignment with other ov-serpins. The physiological target protease is unknown and the in vitro translated hurpin does not form SDS-stable complexes with a variety of known serine proteases. Expression of hurpin is restricted to epidermal cells where two distinct transcripts of 3.0 and 3.4 kb are detectable. Furthermore, expression of hurpin appears to be related to the activation or proliferation state of keratinocytes, since hurpin transcripts are more abundant in immortalized keratinocytes (HaCaT) and in cultured normal human keratinocytes, compared to the expression in normal skin. Moreover, in psoriasis, a skin disease characterized by hyperproliferation of keratinocytes and responsive to therapeutic UV irradiation, overexpression of hurpin is noted in psoriatic skin lesions compared to non-lesional skin.
Asunto(s)
Inhibidores de Serina Proteinasa/genética , Serpinas/genética , Piel/metabolismo , Secuencia de Aminoácidos , Antígenos de Neoplasias/química , Sitios de Unión , Línea Celular , Clonación Molecular , Humanos , Queratinocitos , Datos de Secuencia Molecular , Ovalbúmina/genética , Psoriasis/metabolismo , Retina/metabolismo , Homología de Secuencia de Aminoácido , Inhibidores de Serina Proteinasa/química , Serpinas/química , Rayos UltravioletaRESUMEN
N-(trifluoromethylphenyl)-2-cyano-3-hydroxy-crotonic acid amide (A77 1726), the physiologically active metabolite of leflunomide, has been described to exert antiproliferative effects in vitro and anti-inflammatory actions in several animal models. Currently, its use is being evaluated in clinical trials in psoriasis, which is characterized by epidermal hyperproliferation and infiltration of inflammatory cells. We studied the effects of A77 1726 on growth and gene expression in cultured epidermal cells by 5-bromo-2'-deoxy-uridine (BrdU) incorporation, reverse transcriptase-polymerase chain reaction (RT-PCR), Northern blot hybridizations and flow cytometry. A77 1726 inhibited epidermal proliferation at concentrations above 5 microM after 24 hr. However, the cells were still fully viable at a concentration of 100 microM. The drug caused a dose-dependent reduction in the mRNA level of the type A receptor for the proinflammatory cytokine interleukin-8 (IL-8-RA) and, in contrast, induced gene expression of the receptor for the anti-inflammatory cytokine IL-10 (IL-10R) at the mRNA and protein levels. In addition, the mRNA and protein levels of the p53 gene, which is a negative cell cycle regulator, were up-regulated by A77 1726. These data suggest that A77 1726 exerts its anti-inflammatory action via the modulation of epidermal gene expression.
Asunto(s)
Compuestos de Anilina/farmacología , Antiinflamatorios no Esteroideos/farmacología , Antígenos CD/genética , Regulación de la Expresión Génica/efectos de los fármacos , Hidroxibutiratos/farmacología , Receptores de Interleucina/genética , Transcripción Genética/efectos de los fármacos , División Celular/efectos de los fármacos , Línea Celular Transformada , Supervivencia Celular/efectos de los fármacos , Crotonatos , Genes p53/efectos de los fármacos , Humanos , Isoxazoles/farmacocinética , Isoxazoles/farmacología , Leflunamida , Nitrilos , ARN Mensajero/genética , Receptores de Interleucina-10 , Receptores de Interleucina-8A , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Toluidinas , Proteína p53 Supresora de Tumor/genéticaRESUMEN
Ultraviolet (UV) light is the most important environmental insult to skin. Even a single exposure to UVB radiation can result in inflammation and may also lead to DNA damage and apoptosis in the acute response of the cutaneous tissue. To elucidate the complex alterations of gene expression in human keratinocytes underlying these UV responses we took advantage of differential display polymerase chain reaction (DD-PCR) technology's ability to detect qualitative and quantitative changes in gene expression in more than two cell populations simultaneously. We demonstrate that low-dose UVB (100 Jm-2) leads to both induction and downregulation of different genes during the 24 h after irradiation in a time-dependent manner. In addition to the identification of known genes as possible effectors or targets in the UV response of human keratinocytes, we here identify a new sequence that is negatively regulated by UVB irradiation and was termed HUR 7 (HaCaT UV repressed). In general our results showed that DD-PCR is a useful tool in the analysis of quantitative changes of mRNA levels in human keratinocytes after UV irradiation. The identification of new UVB-repressed genes offers the opportunity to identify unrecognized molecular mechanisms in the UV response of human cells.
Asunto(s)
Expresión Génica/efectos de la radiación , Queratinocitos/efectos de la radiación , Reacción en Cadena de la Polimerasa/métodos , ARN Mensajero/metabolismo , Rayos Ultravioleta , Northern Blotting , Células Cultivadas , ADN Complementario/química , Humanos , Datos de Secuencia Molecular , Análisis de Secuencia de ADNRESUMEN
The chronic skin disease psoriasis is characterized by epidermal hyperproliferation and inflammation. The exact etiology of the disease is still unknown. At the molecular level, overexpression of growth factors and proinflammatory cytokines such as IL-8 and the corresponding receptor has been described in psoriatic plaques. On the other hand, the loss of inhibitory control mechanisms is involved in the pathogenesis of the disease, as exemplified by the reduced mRNA levels for the cell cycle inhibitor p53 found in lesional skin. Here we extend these findings to a cytokine with negative regulatory functions, IL-10. Only under certain conditions are human keratinocytes able to synthesize IL-10. In skin, pathological overexpression of IL-10 was described om atopic dermatitis. IL-10 exerts its effects via a specific receptor (IL-10R). We show here for the first time the presence and functionality of IL-10R in epidermal cells and its dramatically decreased expression in acute exanthematic psoriatic epidermis by in vitro and in situ binding studies. These results were substantiated using semiquantitative reverse transcriptase-PCR, demonstrating decreased expression of the IL-10R gene in psoriatic skin, its down-modulation by the proinflammatory cytokine IL-8, and its pharmacological induction in cultured cells. Biological responsiveness of epidermal cells toward IL-10 could also be demonstrated by a reduction of the growth rate and inhibition of IFN-gamma-induced HLA-DR expression. Our results provide the first evidence for a role of the IL-10R gene in the homeostasis of the epidermis and substantiate the concept of a loss of negative regulatory peptides as a step in the eruption of psoriasis.