RESUMEN
Disordered proteins have long been known to help mediate tolerance to different abiotic stresses including freezing, osmotic stress, high temperatures, and desiccation in a diverse set of organisms. Recently, three novel families of intrinsically disordered proteins were identified in tardigrades, microscopic animals capable of surviving a battery of environmental extremes. These three families include the Cytoplasmic-, Secreted-, and Mitochondrial- Abundant Heat Soluble (CAHS, SAHS, and MAHS) proteins, which are collectively termed Tardigrade Disordered Proteins (TDPs). At the level of sequence conservation TDPs are unique to tardigrades, and beyond their high degree of disorder the CAHS, SAHS, and MAHS families do not resemble one another. All three families are either highly expressed constitutively, or significantly enriched in response to desiccation. In vivo, ex vivo, and in vitro experiments indicate functional roles for members of each TDP family in mitigating cellular perturbations induced by various abiotic stresses. What is currently lacking is a comprehensive and holistic understanding of the fundamental mechanisms by which TDPs function, and the properties of TDPs that allow them to function via those mechanisms. A quantitative and systematic approach is needed to identify precisely what cellular damage TDPs work to prevent, what sequence features are important for these functions, and how those sequence features contribute to the underlying mechanisms of protection. Such an approach will inform us not only about these fascinating proteins, but will also provide insights into how the sequence of a disordered protein can dictate its functional, structural, and dynamic properties. Video Abstract.
Asunto(s)
Adaptación Fisiológica , Proteínas Intrínsecamente Desordenadas/metabolismo , Estrés Fisiológico , Tardigrada/fisiología , Secuencia de Aminoácidos , Animales , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/metabolismo , Proteínas Intrínsecamente Desordenadas/química , Proteínas Mitocondriales/química , Proteínas Mitocondriales/metabolismoRESUMEN
A new diarylheptanoid containing a chalcone moiety, katsumain H (1), was isolated from the seeds of Alpinia katsumadai. The structure was elucidated using a combination of 1D/2D NMR spectroscopy and mass spectrometry data analysis. The absolute configurations of C-3, C-5, and C-7 in 1 were assigned based on its optical rotation and after comparing its NMR chemical shifts with those of its diastereoisomers, katsumain E and katsumain F, which were previously isolated from this plant and characterized. In this study, the stimulatory effects of compounds 1 and 2 were evaluated on heat shock factor 1 (HSF1), heat shock protein 27 (HSP27), and HSP70. Compounds 1 and 2 increased the expression of HSF1 (1.056- and 1.200-fold, respectively), HSP27 (1.312- and 1.242-fold, respectively), and HSP70 (1.234- and 1.271-fold, respectively), without increased cytotoxicity.
Asunto(s)
Alpinia/química , Chalcona/química , Diarilheptanoides/química , Proteínas de Choque Térmico/química , Espectroscopía de Resonancia Magnética , Espectrometría de Masas , Estructura Molecular , Extractos Vegetales/química , Semillas/químicaRESUMEN
Vertebrate lens is one of the tissues with the highest soluble protein concentration. The predominant soluble proteins in lens fiber cells are crystallins, and among them, α-crystallins belong to the small heat shock protein family with chaperone-like activity. Although α-crystallins are highly soluble in waters, α-crystallins have been detected in the membrane-bound fraction of lens, which will increase in the aged or cataractous lens. In this research, we found αA-crystallin exhibited a complex thermal transition with remarkable changes in secondary and quaternary structures. Treatment of αA-crystallin at high temperatures induced larger oliogomers with higher hydrophobic exposure. Both heat-treated and untreated αA-crystallin could insert into lipid monolayer directly as revealed by monolayer surface pressure experiments. Heat-treatment facilitated the membrane insertion of αA-crystallin and increased the membrane-bound fraction in the cells. The membrane-binding ability of αA-crystallin could be altered by cataract-causing mutations R116C, R116H and Y118D. Our results suggested that the irreversible changes in oligomer size induced by various stresses might promote the membrane association of αA-crystallin and therefore might play a role in aged cataract. Alternations in the membrane binding ability of α-crystallins might be important to the understanding of both aged and congenital cataracts.
Asunto(s)
Membrana Celular/química , Cristalinas/química , 1,2-Dipalmitoilfosfatidilcolina/química , Animales , Catarata/metabolismo , Bovinos , Cromatografía , ADN Complementario/metabolismo , Células HeLa , Proteínas de Choque Térmico/química , Humanos , Lípidos/química , Microscopía Fluorescente , Mutación , Fosfatidilserinas/química , Presión , Unión Proteica , Estructura Cuaternaria de Proteína , Estructura Secundaria de Proteína , Albúmina Sérica Bovina/química , TemperaturaRESUMEN
A growing body of evidence from several laboratories points at nonmechanical functions of keratin intermediate filaments (IF), such as control of apoptosis, modulation of signaling, or regulation of innate immunity, among others. While these functions are generally assigned to the ability of IF to scaffold other proteins, direct mechanistic causal relationships between filamentous keratins and the observed effects of keratin knockout or mutations are still missing. We have proposed that the scaffolding of chaperones such as Hsp70/40 may be key to understand some IF nonmechanical functions if unique features or specificity of the chaperoning activity in the IF scaffold can be demonstrated. The same criteria of uniqueness could be applied to other biochemical functions of the IF scaffold. Here, we describe a subcellular fractionation technique based on established methods of keratin purification. The resulting keratin-enriched fraction contains several proteins tightly associated with the IF scaffold, including Hsp70/40 chaperones. Being nondenaturing, this fractionation method enables direct testing of chaperoning and other enzymatic activities associated with IF, as well as supplementation experiments to determine the need for soluble (cytosolic) proteins. This method also permits to analyze inhibitory activity of cytosolic proteins at independently characterized physiological concentrations. When used as complementary approaches to knockout, knockdown, or site-directed mutagenesis, these techniques are expected to shed light on molecular mechanisms involved in the effects of IF loss of function.
Asunto(s)
Proteínas de Choque Térmico/química , Queratinas Específicas del Pelo/química , Proteína Quinasa C/química , Animales , Células CACO-2 , Fraccionamiento Celular , Humanos , Filamentos Intermedios/enzimología , Mucosa Intestinal/citología , Ratones , Fosforilación , Pliegue de Proteína , Procesamiento Proteico-PostraduccionalRESUMEN
Since the initial discovery of the protective role of heat shock protein (HSP) 60 in arthritis, T cell recognition of endogenous HSP was found to be one of the possible underlying mechanisms. Recently we have uncovered potent disease-suppressive Tregs (anti-inflammatory immunosuppressive T cells) recognizing HSP70 self-antigens, and enabling selective targeting of such Tregs to inflamed tissues. HSP70 is a major contributor to the major histocompatibility complex (MHC) Class II ligandome and we have shown that a conserved HSP70-epitope (B29) is abundantly present in murine MHC Class II. Upon transfer, B29-induced CD4+CD25+Foxp3+T cells suppressed established proteoglycan-induced arthritis (PGIA) in mice. These self-antigen specific Tregs were activated in vivo and as little as 4.000 cells sufficed to fully inhibit arthritis. Furthermore, in vivo depletion of transferred Tregs abrogated disease suppression. Given that B29 can be presented by most human MHC class II molecules and that B29 inhibited arthritis in HLA-DQ8 (human MHC) transgenic mice, we feel that therapeutic vaccination with selected HSP peptides can be an effective route for induction of anti-inflammatory Tregs as a novel intervention in chronic inflammatory diseases.
Asunto(s)
Proteínas de Choque Térmico/inmunología , Inflamación/inmunología , Linfocitos T Reguladores/inmunología , Secuencia de Aminoácidos , Animales , Artritis/inmunología , Artritis/metabolismo , Epítopos/inmunología , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/metabolismo , Humanos , Inflamación/metabolismo , Mediadores de Inflamación/inmunología , Mediadores de Inflamación/metabolismo , Datos de Secuencia Molecular , Linfocitos T Reguladores/metabolismoRESUMEN
BACKGROUND: Increased intake of ω-3 long-chain polyunsaturated fatty acids (LCPUFAs) and use of peroxisome proliferator activator receptor (PPAR)-activating drugs are associated with attenuation of pathologic retinal angiogenesis. ω-3 LCPUFAs are endogenous agonists of PPARs. We postulated that DNA sequence variation in PPAR gamma (PPARG) co-activator 1 alpha (PPARGC1A), a gene encoding a co-activator of the LCPUFA-sensing PPARG-retinoid X receptor (RXR) transcription complex, may influence neovascularization (NV) in age-related macular degeneration (AMD). METHODS: We applied exact testing methods to examine distributions of DNA sequence variants in PPARGC1A for association with NV AMD and interaction of AMD-associated loci in genes of complement, lipid metabolism, and VEGF signaling systems. Our sample contained 1858 people from 3 elderly cohorts of western European ancestry. We concurrently investigated retinal gene expression profiles in 17-day-old neonatal mice on a 2% LCPUFA feeding paradigm to identify LCPUFA-regulated genes both associated with pathologic retinal angiogenesis and known to interact with PPARs or PPARGC1A. RESULTS: A DNA coding variant (rs3736265) and a 3'UTR-resident regulatory variant (rs3774923) in PPARGC1A were independently associated with NV AMD (exact P = 0.003, both SNPs). SNP-SNP interactions existed for NV AMD (P<0.005) with rs3736265 and a AMD-associated variant in complement factor B (CFB, rs512559). PPARGC1A influences activation of the AMD-associated complement component 3 (C3) promoter fragment and CFB influences activation and proteolysis of C3. We observed interaction (P ≤ 0.003) of rs3736265 with a variant in vascular endothelial growth factor A (VEGFA, rs3025033), a key molecule in retinal angiogenesis. Another PPARGC1A coding variant (rs8192678) showed statistical interaction with a SNP in the VEGFA receptor fms-related tyrosine kinase 1 (FLT1, rs10507386; P ≤ 0.003). C3 expression was down-regulated 2-fold in retinas of ω-3 LCPUFA-fed mice - these animals also showed 70% reduction in retinal NV (P ≤ 0.001). CONCLUSION: Ligands and co-activators of the ω-3 LCPUFA sensing PPAR-RXR axis may influence retinal angiogenesis in NV AMD via the complement and VEGF signaling systems. We have linked the co-activator of a lipid-sensing transcription factor (PPARG co-activator 1 alpha, PPARGC1A) to age-related macular degeneration (AMD) and AMD-associated genes.
Asunto(s)
Proteínas del Sistema Complemento/genética , Proteínas de Choque Térmico/genética , Degeneración Macular/genética , Degeneración Macular/metabolismo , Neovascularización Patológica/genética , Transducción de Señal , Factores de Transcripción/genética , Factor A de Crecimiento Endotelial Vascular/metabolismo , Anciano , Anciano de 80 o más Años , Animales , Estudios de Cohortes , Ácidos Grasos Omega-3/metabolismo , Femenino , Perfilación de la Expresión Génica , Redes Reguladoras de Genes , Proteínas de Choque Térmico/química , Humanos , Degeneración Macular/patología , Masculino , Ratones , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma , Polimorfismo de Nucleótido Simple , Retina/metabolismo , Retina/patología , Factores de Transcripción/química , Factor A de Crecimiento Endotelial Vascular/genéticaRESUMEN
Heat shock proteins (HSPs) play various stress-protective roles in plants. In this study, three HSP genes were isolated from a suppression subtractive hybridization (SSH) cDNA library of Ginkgo biloba leaves treated with cold stress. Based on the molecular weight, the three genes were designated GbHSP16.8, GbHSP17 and GbHSP70. The full length of the three genes were predicted to encode three polypeptide chains containing 149 amino acids (Aa), 152 Aa, and 657 Aa, and their corresponding molecular weights were predicted as follows: 16.67 kDa, 17.39 kDa, and 71.81 kDa respectively. The three genes exhibited distinctive expression patterns in different organs or development stages. GbHSP16.8 and GbHSP70 showed high expression levels in leaves and a low level in gynoecia, GbHSP17 showed a higher transcription in stamens and lower level in fruit. This result indicates that GbHSP16.8 and GbHSP70 may play important roles in Ginkgo leaf development and photosynthesis, and GbHSP17 may play a positive role in pollen maturation. All three GbHSPs were up-regulated under cold stress, whereas extreme heat stress only caused up-regulation of GbHSP70, UV-B treatment resulted in up-regulation of GbHSP16.8 and GbHSP17, wounding treatment resulted in up-regulation of GbHSP16.8 and GbHSP70, and abscisic acid (ABA) treatment caused up-regulation of GbHSP70 primarily.
Asunto(s)
Regulación de la Expresión Génica de las Plantas , Ginkgo biloba/genética , Ginkgo biloba/fisiología , Proteínas de Choque Térmico/genética , Proteínas de Plantas/genética , Secuencia de Aminoácidos , Frío , Ginkgo biloba/química , Proteínas de Choque Térmico/química , Datos de Secuencia Molecular , Proteínas de Plantas/química , Alineación de Secuencia , Estrés FisiológicoRESUMEN
Bioactive compounds from the medicinal plant, Eurycoma longifolia Jack have been shown to promote anti-proliferative effects on various cancer cell lines. Here we examined the effects of purified eurycomanone, a quassinoid found in Eurycoma longifolia Jack extract, on the expression of selected genes of the A549 lung cancer cells. Eurycomanone inhibited A549 lung cancer cell proliferation in a dose-dependent manner at concentrations ranging from 5 to 20 µg/ml. The concentration that inhibited 50% of cell growth (GI(50)) was 5.1 µg/ml. The anti-proliferative effects were not fully reversible following the removal of eurycomanone, in which 30% of cell inhibition still remained (p<0.0001, T-test). At 8 µg/ml (GI(70)), eurycomanone suppressed anchorage-independent growth of A549 cells by >25% (p<0.05, T-test, n=8) as determined using soft agar colony formation assay. Cisplatin, a chemotherapy drug used for the treatment of non small cell lung cancer on the other hand, inhibited A549 cells proliferation at concentrations ranging from 0.2 µg/ml to 15 µg/ml with a GI(50) of 0.58 µg/ml. The treatment with eurycomanone reduced the abundance expression of the lung cancer markers, heterogeneous nuclear ribonucleoprotein (hnRNP) A2/B1, p53 tumor suppressor protein and other cancer-associated genes including prohibitin (PHB), annexin 1 (ANX1) and endoplasmic reticulum protein 28 (ERp28) but not the house keeping genes. The mRNA expressions of all genes with the exception of PHB were significantly downregulated, 72 h after treatment (p<0.05, T-test, n=9). These findings suggest that eurycomanone at viable therapeutic concentrations of 5-20 µg/ml exhibited significant anti-proliferative and anti-clonogenic cell growth effects on A549 lung cancer cells. The treatment also resulted in suppression of the lung cancer cell tumor markers and several known cancer cell growth-associated genes.
Asunto(s)
Anexina A1/química , Biomarcadores de Tumor/química , Proteínas de Choque Térmico/química , Neoplasias Pulmonares/tratamiento farmacológico , Extractos Vegetales/farmacología , Cuassinas/farmacología , Proteínas Represoras/química , Antineoplásicos Fitogénicos/química , Antineoplásicos Fitogénicos/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Cisplatino/farmacología , Relación Dosis-Respuesta a Droga , Electroforesis en Gel Bidimensional , Eurycoma/química , Humanos , Immunoblotting , Neoplasias Pulmonares/química , Extractos Vegetales/química , Extractos Vegetales/aislamiento & purificación , Raíces de Plantas/química , Prohibitinas , Cuassinas/química , Cuassinas/aislamiento & purificación , Ensayo de Tumor de Célula Madre , Proteína p53 Supresora de Tumor/químicaRESUMEN
Binding of heat shock factors (Hsfs) to heat shock elements (HSEs) leads to transcriptional regulation of heat shock genes. Genome-wide, 953 rice genes contain perfect-type, 695 genes gap-type and 1584 genes step-type HSE sequences in their 1-kb promoter region. The rice genome contains 13 class A, eight class B and four class C Hsfs (OsHsfs) and has OsHsf26 (which is of variant type) genes. Chemical cross-linking analysis of in vitro synthesized OsHsf polypeptides showed formation of homotrimers of OsHsfA2c, OsHsfA9 and OsHsfB4b proteins. Binding analysis of polypeptides with oligonucleotide probes containing perfect-, gap-, and step-type HSE sequences showed that OsHsfA2c, OsHsfA9 and OsHsfB4b differentially recognize various model HSEs as a function of varying reaction temperatures. The homomeric form of OsHsfA2c and OsHsfB4b proteins was further noted by the bimolecular fluorescence complementation approach in onion epidermal cells. In yeast two-hybrid assays, OsHsfB4b showed homomeric interaction as well as distinct heteromeric interactions with OsHsfA2a, OsHsfA7, OsHsfB4c and OsHsf26. Transactivation activity was noted in OsHsfA2c, OsHsfA2d, OsHsfA9, OsHsfC1a and OsHsfC1b in yeast cells. These differential patterns pertaining to binding with HSEs and protein-protein interactions may have a bearing on the cellular functioning of OsHsfs under a range of different physiological and environmental conditions.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteínas de Choque Térmico/metabolismo , Oryza/metabolismo , Proteínas de Plantas/metabolismo , Elementos de Respuesta , Factores de Transcripción/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Células Cultivadas , Reactivos de Enlaces Cruzados/química , ADN de Plantas/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Ensayo de Cambio de Movilidad Electroforética , Genes de Plantas , Factores de Transcripción del Choque Térmico , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/genética , Respuesta al Choque Térmico , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Cebollas/citología , Cebollas/metabolismo , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Epidermis de la Planta/citología , Epidermis de la Planta/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Factores de Transcripción/química , Factores de Transcripción/genética , Activación Transcripcional , Técnicas del Sistema de Dos HíbridosRESUMEN
Endoplasmic reticulum (ER) stress has recently been implicated as a cause for obesity-related insulin resistance; however, what causes ER stress in obesity has remained uncertain. Here, we have tested the hypothesis that macronutrients can cause acute (ER) stress in rat liver. Examined were the effects of intravenously infused glucose and/or lipids on proximal ER stress sensor activation (PERK, eIF2-α, ATF4, Xbox protein 1 (XBP1s)), unfolded protein response (UPR) proteins (GRP78, calnexin, calreticulin, protein disulphide isomerase (PDI), stress kinases (JNK, p38 MAPK) and insulin signaling (insulin/receptor substrate (IRS) 1/2 associated phosphoinositol-3-kinase (PI3K)) in rat liver. Glucose and/or lipid infusions, ranging from 23.8 to 69.5 kJ/4 h (equivalent to between ~17% and ~50% of normal daily energy intake), activated the proximal ER stress sensor PERK and ATF6 increased the protein abundance of calnexin, calreticulin and PDI and increased two GRP78 isoforms. Glucose and glucose plus lipid infusions induced comparable degrees of ER stress, but only infusions containing lipid activated stress kinases (JNK and p38 MAPK) and inhibited insulin signaling (PI3K). In summary, physiologic amounts of both glucose and lipids acutely increased ER stress in livers 12-h fasted rats and dependent on the presence of fat, caused insulin resistance. We conclude that this type of acute ER stress is likely to occur during normal daily nutrient intake.
Asunto(s)
Retículo Endoplásmico/metabolismo , Emulsiones Grasas Intravenosas/administración & dosificación , Glucosa/administración & dosificación , Hígado/metabolismo , Fosfolípidos/administración & dosificación , Aceite de Soja/administración & dosificación , Estrés Fisiológico , Factor de Transcripción Activador 6/metabolismo , Animales , Emulsiones/administración & dosificación , Emulsiones/efectos adversos , Chaperón BiP del Retículo Endoplásmico , Emulsiones Grasas Intravenosas/efectos adversos , Glucosa/efectos adversos , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/metabolismo , Infusiones Intravenosas , Insulina/administración & dosificación , Insulina/sangre , Insulina/metabolismo , Resistencia a la Insulina , Cinética , Sistema de Señalización de MAP Quinasas , Masculino , Fosfatidilinositol 3-Quinasa/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Fosfolípidos/efectos adversos , Ratas , Ratas Sprague-Dawley , Aceite de Soja/efectos adversos , Respuesta de Proteína Desplegada , eIF-2 Quinasa/metabolismoRESUMEN
The small heat shock protein SsHSP14.1 from the hyper-thermophilic archeaon, Sulfolobus solfataricus (S. solfataricus) was able to protect proteins from thermal aggregation and prevent enzymes from heat induced inactivation. According to the 3D (dimensional) structural model of SsHSP14.1 developed by us before, the region L5-7 (ß5-ß7, 68-82 residues) plays an important role for the oligomerization of SsHSP14.1 and its chaperone function. Here, to validate the findings, an in-depth investigation was conducted of both the wild type SsHSP14.1 and its deletion mutant DEL75-79. With E. coli proteins and bromelain as substrate, the deletion mutant DEL75-79 can protect them from thermo-aggregating as effective as the wild protein. Interestingly, unlike the wild protein, DEL75-79 was unable to prevent bromelain and EcoRI from thermo-inactivating. Results of size exclusion HPLC showed that the oligomerization state was changed in mutant protein. This was in accordance with the changed structure and lower hydrophobicity of DEL75-79. These outcomes proved that the L5-7 loop did play a role for the oligomerizing SsHSP14.1, and that the residues 75-79 were indispensable for its function of prevent enzymes from thermo-inactivating.
Asunto(s)
Proteínas Arqueales/química , Proteínas Arqueales/metabolismo , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/metabolismo , Sulfolobus solfataricus , Biotecnología , Interacciones Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Multimerización de Proteína , Estructura Cuaternaria de ProteínaRESUMEN
The recent genetic and biochemical studies reveal a considerable overlap among cellular processes in response to heat and oxidative stress stimuli in plants suggesting an intimate relationship between the heat-shock response and oxidative stress responses. Pennisetum glaucum (Pg) seedlings were exposed to heat stress (42 degrees C for 0.5, 1.0 and 24h) and a mixture of RNA from all the heat stressed seedlings was used to prepare cDNA. Full-length cDNA clones encoding for cytoplasmic ascorbate peroxidase 1 (PgAPX1) and heat-shock factor (PgHSF) were isolated by screening heat stress-specific cDNA library using corresponding EST sequences as radioactive probes. These full-length cDNAs were expressed in E. coli and their recombinant proteins were purified to near homogeneity. The recombinant PgAPX1 preferred ascorbate but did not accept guaiacol as a reducing substrate. Over-expression of PgAPX1 protects E. coli cells against methyl viologen-induced oxidative stress. Sequence analysis of PgAPX1 promoter identified a number of putative stress regulatory cis-elements including a heat-shock element (HSE). Heat-shock transcription factors (HSFs) play a central role in mediating these overlapping cellular processes. Gel shift analysis and competition with specific and non-specific unlabeled DNA probes showed a specific interaction between HSE of PgAPX1 and the PgHSF protein. Expression analysis of PgHSF in Pennisetum showed maximum increase in transcript level in response to heat stress within 30 min of exposure and slowed down at subsequent time points of heat stress, indicating a typical characteristic of HSF in terms of early responsiveness. Expression of PgAPX1 significantly increased under heat-stress condition; however, the maximum expression observed at 24h of heat stress. In gel activity of PgAPX1 in Pennisetum plants also showed an increase in response to heat stress (42 degrees C) being maximum at 24h and these trends are in conformity with the expression pattern of PgAPX1. Expression patterns and interactive specificity of HSF with HSE (PgAPX1) suggest a probable vital interlink in heat and oxidative stress signaling pathways that plays a significant role in comprehending the underlying mechanisms in plant abiotic stress tolerance.
Asunto(s)
Proteínas de Unión al ADN/genética , Proteínas de Choque Térmico/genética , Respuesta al Choque Térmico/genética , Estrés Oxidativo/genética , Pennisetum/genética , Peroxidasas/genética , Proteínas de Plantas/genética , Factores de Transcripción/genética , Secuencia de Aminoácidos , Ascorbato Peroxidasas , Secuencia de Bases , Clonación Molecular , ADN Complementario/química , Proteínas de Unión al ADN/química , Escherichia coli/genética , Regulación de la Expresión Génica de las Plantas , Biblioteca de Genes , Factores de Transcripción del Choque Térmico , Proteínas de Choque Térmico/química , Datos de Secuencia Molecular , Pennisetum/enzimología , Peroxidasas/química , Filogenia , Proteínas de Plantas/química , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Temperatura , Factores de Transcripción/químicaRESUMEN
HspA, a member of the GroES chaperonin family, is a small protein found in Helicobacter pylori with a unique histidine- and cysteine-rich domain at the C terminus. In this work, we overexpressed, purified, and characterized this protein both in vitro and in vivo. The apo form of the protein binds 2.10 +/- 0.07 Ni(2+) or 1.98 +/- 0.08 Bi(3+) ions/monomer with a dissociation constant (K(d)) of 1.1 or 5.9 x 10(-19) microm, respectively. Importantly, Ni(2+) can reversibly bind to the protein, as the bound nickel can be released either in the presence of a chelating ligand, e.g. EDTA, or at an acidic pH (pH((1/2)) 3.8 +/- 0.2). In contrast, Bi(3+) binds almost irreversibly to the protein. Both gel filtration chromatography and native electrophoresis demonstrated that apo-HspA exists as a heptamer in solution. Unexpectedly, binding of Bi(3+) to the protein altered its quaternary structure from a heptamer to a dimer, indicating that bismuth may interfere with the biological functions of HspA. When cultured in Ni(2+)-supplemented M9 minimal medium, Escherichia coli BL21(DE3) cells expressing wild-type HspA or the C-terminal deletion mutant clearly indicated that the C terminus might protect cells from high concentrations of external Ni(2+). However, an opposite phenomenon was observed when the same E. coli hosts were grown in Bi(3+)-supplemented medium. HspA may therefore play a dual role: to facilitate nickel acquisition by donating Ni(2+) to appropriate proteins in a nickel-deficient environment and to carry out detoxification via sequestration of excess nickel. Meanwhile, HspA can be a potential target of the bismuth antiulcer drug against H. pylori.
Asunto(s)
Proteínas Bacterianas/química , Bismuto/química , Chaperoninas/química , Proteínas de Choque Térmico/química , Helicobacter pylori/química , Metaloproteínas/química , Níquel/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , Proteínas Bacterianas/metabolismo , Bismuto/uso terapéutico , Chaperoninas/genética , Chaperoninas/aislamiento & purificación , Chaperoninas/metabolismo , Dimerización , Ácido Edético/química , Escherichia coli/crecimiento & desarrollo , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/aislamiento & purificación , Proteínas de Choque Térmico/metabolismo , Infecciones por Helicobacter/tratamiento farmacológico , Infecciones por Helicobacter/genética , Infecciones por Helicobacter/metabolismo , Helicobacter pylori/genética , Helicobacter pylori/metabolismo , Histidina/química , Histidina/genética , Histidina/metabolismo , Concentración de Iones de Hidrógeno , Metaloproteínas/genética , Metaloproteínas/aislamiento & purificación , Metaloproteínas/metabolismo , Níquel/metabolismo , Níquel/farmacología , Unión Proteica/fisiología , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína/fisiología , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismoRESUMEN
In Saccharomyces cerevisiae, the intracellular concentration of trehalose increases rapidly in response to many environmental stresses, including heat shock. These high trehalose levels have been correlated with tolerance to adverse conditions and led to the model that trehalose functions as a chemical cochaperone. Here, we show that the transcriptional activity of Hsf1 during the heat shock response depends on trehalose. Strains with low levels of trehalose have a diminished transcriptional response to heat shock, while strains with high levels of trehalose have an enhanced transcriptional response to heat shock. The enhanced transcriptional response does not require the other heat-responsive transcription factors Msn2/4 but is dependent upon heat and Hsf1. In addition, the phosphorylation levels of Hsf1 correlate with both transcriptional activity and the presence of trehalose. These in vivo results support a new role for trehalose, where trehalose directly modifies the dynamic range of Hsf1 activity and therefore influences heat shock protein mRNA levels in response to stress.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteínas de Choque Térmico/metabolismo , Respuesta al Choque Térmico , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Factores de Transcripción/metabolismo , Trehalosa/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Regulación Fúngica de la Expresión Génica , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/genética , Hipertermia Inducida , Fosforilación , Estructura Terciaria de Proteína , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Factores de Transcripción/química , Factores de Transcripción/genética , Transcripción Genética , Trehalosa/análisisRESUMEN
A cDNA clone was isolated after difference screening from cotyledons of two-week cold-treated Ammopiptanthus mongolicus. The full-length cDNA sequence [designated as AmCIP (A. mongolicus cold-induced protein) gene] was 806 bp long and contained a 465 bp open reading frame (ORF) encoding a 16.6 kD protein of 154 amino acids. Bioinformatic analyses indicated that CIP belongs to dehydrin family with the features of high hydrophilicity, a helix K-segment, a long Gly-rich region and a phosphorylatable tract of Ser as well as deficiency in Cys and Trp. The expression of CIP gene increased after two weeks of cold treatment and more expression was detected in radicle than in cotyledon. And PCR amplification of the AmCIP gene from genome of A. mongolicus revealed this gene has no intron. Function prediction suggested this protein seems to protect the stabilization of membrane structure and prevent macromolecular coagulation or sequestrate calcium ions by association or disassociation with membrane under low temperature conditions.
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Frío , Fabaceae/genética , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/genética , Proteínas de Plantas/química , Proteínas de Plantas/genética , Secuencia de Aminoácidos , Secuencia de Bases , ADN Complementario/genética , Fabaceae/fisiología , Proteínas de Choque Térmico/fisiología , Datos de Secuencia Molecular , Proteínas de Plantas/fisiologíaRESUMEN
Seventeen mutants with one, two or three amino acids substitutions of human protein p14.5, homologue to well-known tumor antigen from goat liver UK114 and a member of proteins YER057c/YIL051c/YjgF family, have been used for structure-functional relation studies and ligand binding analysis using cross-linking by triacryloyl-hexahydro-s-triazine (TAT), size exclusion chromatography, free fatty acid and 8-anilino-1-naphthalenesulfonic acid (ANS) binding assays. Amino acids having the most significant impact on the ligand binding activity have been determined: R107, N93, Y21 and F89. Arginine 107 has been identified as the most accessible amino acid in the cleft. Trimeric structure of protein p14.5 has been confirmed as being essential for stoichiometric small ligand binding activity and oligomeric structure of p14. Ligand binding activity may be related with the biological functions of these proteins, which still are not understood well.
Asunto(s)
Proteínas de Escherichia coli/química , Proteínas de Choque Térmico/química , Proteínas Mitocondriales/química , Ribonucleasas/química , Proteínas de Saccharomyces cerevisiae/química , Naftalenosulfonatos de Anilina/farmacología , Sitios de Unión , Bioquímica/métodos , Cromatografía , Clonación Molecular , Reactivos de Enlaces Cruzados/farmacología , ADN Complementario/metabolismo , Electroforesis en Gel de Poliacrilamida , Ácidos Grasos/química , Colorantes Fluorescentes/farmacología , Humanos , Cinética , Ligandos , Modelos Moleculares , Conformación Molecular , Mutagénesis Sitio-Dirigida , Mutación , Unión Proteica , Relación Estructura-Actividad , Triazinas/farmacologíaRESUMEN
Peroxisome proliferator-activated receptor gamma coactivator 1alpha (PGC-1alpha), a tissue-specific and inducible transcriptional coactivator for several nuclear receptors, plays a key role in energy metabolism. We report here that PGC-1alpha coactivator activity is potentiated by arginine methylation by protein arginine methyltransferase 1 (PRMT1), another nuclear receptor coactivator. Mutation of three substrate arginines in the C-terminal region of PGC-1alpha abolished the cooperative coactivator function of PGC-1alpha and PRMT1, and compromised the ability of PGC-1alpha to induce endogenous target genes. Finally, endogenous PRMT1 contributes to PGC-1alpha coactivator activity, and to the induction of genes important for mitochondrial biogenesis.
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Proteínas de Choque Térmico/metabolismo , Factores de Transcripción/metabolismo , Animales , Arginina/química , Secuencia de Bases , Línea Celular , ADN Complementario/genética , Regulación de la Expresión Génica , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/genética , Humanos , Técnicas In Vitro , Metilación , Mutagénesis Sitio-Dirigida , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma , Estructura Terciaria de Proteína , Proteína-Arginina N-Metiltransferasas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Factores de Transcripción/química , Factores de Transcripción/genéticaRESUMEN
Tumor development and progression consist of a series of complex processes involving multiple changes in gene expression (Paolo et al. Physiol. Rev., 1993, 73, 161-195; Lance et al. Cell., 1991, 64, 327-336). Tumor cells acquire an invasive and metastatic phenotype that is the main cause of death for cancer patients. Therefore, for early diagnosis and effective therapeutic intervention, we need to detect the alterations associated with transition from benign to malignant tumor cells on a molecular basis. To unravel alterations concerned with tumor progression, the proteomic approach has attracted great attention because it can identify qualitative and quantitative changes in protein composition, including post-translational modifications. In this study, we performed proteomic differential display analysis for the expression of intracellular proteins in the regressive cancer cell line QR-32 and the inflammatory cell-promoting progressive cancer cell line QRsP-11 of murine fibrosarcoma by two-dimensional gel electrophoresis and mass spectrometry using an Agilent 1100 LC/MSD Trap XCT. We found 11 protein spots whose expression was different between QR-32 and QRsP-11 cells and identified nine proteins, seven of which, calreticulin precursor, tropomyosin 1 alpha chain, annexin A5, heat shock protein (HSP)90-alpha, HSP90-beta, PEBP, and Prx II, were over-expressed, and two, Anp32e and HDGF, which were down-regulated. The results suggest an important complementary role for proteomics in identification of molecular abnormalities in tumor progression.
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Perfilación de la Expresión Génica/métodos , Regulación de la Expresión Génica , Neoplasias/metabolismo , Neoplasias/patología , Proteómica/métodos , Animales , Western Blotting , Calreticulina/metabolismo , Línea Celular Tumoral , Cromatografía Líquida de Alta Presión , Cromatografía Liquida , Progresión de la Enfermedad , Electroforesis en Gel Bidimensional , Proteínas HSP90 de Choque Térmico/química , Proteínas de Choque Térmico/química , Procesamiento de Imagen Asistido por Computador , Immunoblotting , Inflamación , Espectrometría de Masas , Ratones , Ratones Endogámicos C57BL , Transducción de SeñalRESUMEN
PURPOSE: Small-molecule growth factor receptor inhibitors block cell growth in vitro and downstream signaling in vivo, but controlled trials in patients with advanced solid tumors have yielded disappointing response rates. To clarify this discrepancy, we compared the patterns of tyrosine phosphoprotein expression in human cancer cells and primary tumors. EXPERIMENTAL DESIGN: Immunoaffinity chromatography, two-dimensional electrophoresis, and antiphosphotyrosine immunoblotting were combined with mass spectrometry to determine the phosphoproteomic signatures of 40 matched normal and malignant tissues from patients with breast or liver cancer. The identities and abundance of the detected tyrosine phosphoproteins were compared with those of ligand-responsive A431 cells. RESULTS: Patterns of tyrosine-phosphorylated proteins are similar among normal tissues of the same origin but vary markedly between different tissues. Primary breast tumors exhibit a strikingly homogeneous tyrosine phosphorylation profile, whereas liver cancers display greater phosphoproteomic diversity. The main breast-tumor-specific tyrosine phosphoproteins are cytoskeletal molecules (actin, tubulin, and vimentin) and molecular chaperones (Hsp70, Hsc71, and Grp75). In contrast, control studies in ligand-stimulated A431 human cancer cells revealed an additional phosphorylated subset of promitogenic phosphoproteins (Grb2, Shc, Jnk2, phospholipase C-gamma, and phosphatidylinositol 3'-kinase). CONCLUSIONS: Identification of cytoskeletal and stress proteins as the most abundant tyrosine phosphoproteins in breast tumors implicates these molecules, rather than promitogenic effectors, as the prime stoichiometric substrates for kinase-inhibitory anticancer drugs in vivo. Because phosphorylated cytoskeletal proteins and chaperones mediate cell motility and apoptotic resistance, respectively, these data raise the intriguing possibility that small-molecule tyrosine kinase inhibitors may be of greatest value either as adjuvant antimetastatic/-invasive drugs or as chemo-/radiosensitizers.
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Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/metabolismo , Quimioterapia Adyuvante/métodos , Citoesqueleto/metabolismo , Tirosina/química , Animales , Apoptosis , Línea Celular Tumoral , Cromatografía/métodos , Electroforesis en Gel Bidimensional/métodos , Proteínas de Choque Térmico/química , Humanos , Immunoblotting , Ligandos , Masculino , Espectrometría de Masas , Ratones , Ratones Endogámicos BALB C , Metástasis de la Neoplasia , Neoplasias/metabolismo , Fosfoproteínas/química , Fosforilación , Fosfotirosina/química , Transducción de SeñalRESUMEN
J-domain proteins are involved in protein importing and sorting, protein translocation into the endoplasmic reticulum and interaction with hsp70, cell cycle regulation and exocytosis. During the large-scale sequencing analysis of a human fetal brain cDNA library, we isolated a cDNA clone encoding a putative 531 amino acid protein with a DnaJ domain and C2H2-type zinc finger motif, which was named as DNAJA5 (human DnaJ homology subfamily A member 5). The putative protein of DNAJA5 gene was homologous to Caenorhabditis elegans DNJ-17 (identity 33.2% similarity 45.1%). By mapping, we located DNAJA5 gene to human chromosome 5p12-5p13. The DNAJA5 gene contained at least 12 exons that spanned more than 25.6 kb of the genomic sequence. The reverse transcription-polymerase chain reaction analysis indicated that it was expressed in the human brain, placenta, kidney and pancreas. DNAJA5 might play a role in co-operating with Hsp70.