Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “ChIP”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 91 records · Page 5Linked to original sources
PubMed1993

Readout of thermoluminescence dosimetry chips using a contact planchet heater.

Thermoluminescence dosimetry (TLD) is performed in many radiotherapy departments using LiF chips which are evaluated by means of a contact heater. The aim of the present study was to investigate the influence of the TL chip position on the heating planchet and to study the self absorption of light in the chips during readout. Experiments were performed using standard LiF chips (3.1 x 3.1 x 0.89 mm3) on a circular heating planchet with a diameter of 13 mm. A two step readout cycle (10s 160 degrees C, 10s 300 degrees C) was used and the integral under the luminescence curve during the second step evaluated. Moving the chip away from the centre of the planchet without adjusting the temperature cycle leads to a peak broadening which can result in a signal loss of up to 40%. The use of a purpose build positioning device tended to improve the reproducibility of the readings. Self absorption of light in a normal chip on the heating planchet was studied by exposing chips in the strong dose gradient of the build up region of a 18 MV X-ray beam. The dose throughout the chip was investigated using a stack of 0.14 mm thick chips. In a 5 x 5 cm2 field the dose varied from 7% at the surface facing the beam to 23% at the opposite side of the chip. Reading the chips with the side facing the beam up in the reader resulted in a 1.9% smaller dose reading than with the chips upside down.(ABSTRACT TRUNCATED AT 250 WORDS)

Thermoluminescent Dosimetry↗
PubMed2005

Protein chip for detection of different HCV antibodies: preparation, quality control, and clinical evaluation.

INTRODUCTION: As a contagious disease caused by hepatitis C virus (HCV) hepatitis C is a serious threat to human health. Therefore, the detection and verification of HCV infection is very important in the treatment of hepatitis C. This study investigated the preparation, quality control, and clinical evaluation of a protein chip capable of simultaneously detecting different HCV antibodies. The aim was to establish a convenient method for the detection of HCV. METHOD: To prepare the protein chip, six antigens including five recombinant HCV antigens (chimeric, core, NS3, NS4, and NS5) and interleukin (IL)-1 were arrayed onto aldehyde-coated slides and blocked using 10% calf serum in phosphate buffered saline. After dilution with sample solution, the serum sample was added to a reaction well on the protein chip. After incubation for 30 minutes at 37 degrees C, fluorescence Cy3-labeled rabbit antihuman IgG was added and incubated again for 30 minutes at 37 degrees C, and then scanned. Positive or negative controls were established from serum samples with or without HCV infection. Clinical evaluation was done by detecting 490 serum samples using the protein chips and ELISA reagents, with 150 of the 490 serum samples confirmed by recombinant immunoblot assay (RIBA). RESULTS: The protein chip for detection of five HCV antibodies was successfully prepared. Fifteen positive controls and 15 negative controls were established as standard samples for quality control. The quality control-passed protein chip was tested again using the standard of the National Institute for the Control of Pharmaceutical and Biological Products (NICPBP), and met the quality control criteria prescribed by the NICPBP. In the clinical evaluation with 490 samples, the coincidence rates between the protein-chip assay and ELISA were 97.4% for positive and 100% for negative results. Five inconsistent samples that were positive in ELISA, but non-positive (four samples) or negative (one) in the protein-chip assay, were confirmed by RIBA (gold standard) to be four non-positive and one negative. The results of 150 samples showed the coincidence rates between protein chip and RIBA were 98.15% for positive and 96.88% for single-segment positive. CONCLUSION: The protein-chip assay has higher sensitivity and specificity than ELISA and has a high coincidence rate with RIBA. The protein chip, characterized by its easy operation and low economic cost, will be very useful for in vitro detection of HCV antibodies.

Hepacivirus↗
PubMed2001

Conductivity detection and quantitation of isotachophoretic analytes on a planar chip with on-line coupled separation channels.

A poly(methylmethacrylate) chip, provided with two separation channels in the column-coupling (CC) arrangement and on-column conductivity detection sensors and intended, mainly, to isotachophoresis (ITP) and ITP-capillary zone electrophoresis (CZE) separations was developed recently. The present work was aimed at assessing its performance relevant to the detection and quantitation of the ITP analytes. Hydrodynamic (HDF) and electroosmotic (EOF) flows of the solution in the separation compartment of the CC chip were suppressed and electrophoresis was a dominant transport process in the ITP separations with model analytes carried out in this context. When the surfaces of the detection electrodes of the conductivity sensors on the chip were appropriately cleaned qualitative indices of the test analytes [relative step heights (RSHs)], provided by a particular detection sensor, agreed within 1% (expressed via RSDs of the RSH values). Their long-term reproducibilities for one sensor, as estimated from 70 ITP runs repeated in 5 days, were 2% or less. Sensor-to-sensor and chip-to-chip fluctuations of the RSH values for the test analytes were 2.5% or less. In addition, experimentally obtained RSH values agreed well with those predicted by the calculations based on the ITP steady-state model. Reproducibilities of the migration velocities attainable on the CC chips with suppressed EOF and HDF, assessed from the migration time measurements of the ITP boundary between well-defined positions on the separation channels of the chips (140 repeated runs on three chips), ranged from 1.4 to 3.3% for the migration times in the range of 100-200 s. Within-day repeatabilities of the time-based zone lengths for the test analytes characterized 2% RSDs, while their day-to-day repeatabilities were less than 5%. Chip-to-chip reproducibilities of the zone lengths, assessed from the data obtained on three chips for 100 ITP runs, were 5-8%.

Electrochemistry↗
PubMed2005

The co-chaperone carboxyl terminus of Hsp70-interacting protein (CHIP) mediates alpha-synuclein degradation decisions between proteasomal and lysosomal pathways.

Alpha-synuclein is a major component of Lewy bodies, the pathological hallmark of Parkinson disease, dementia with Lewy bodies, and related disorders. Misfolding and aggregation of alpha-synuclein is thought to be a critical cofactor in the pathogenesis of certain neurodegenerative diseases. In the current study, we investigate the role of the carboxyl terminus of Hsp70-interacting protein (CHIP) in alpha-synuclein aggregation. We demonstrate that CHIP is a component of Lewy bodies in the human brain, where it colocalizes with alpha-synuclein and Hsp70. In a cell culture model, endogenous CHIP colocalizes with alpha-synuclein and Hsp70 in intracellular inclusions, and overexpression of CHIP inhibits alpha-synuclein inclusion formation and reduces alpha-synuclein protein levels. We demonstrate that CHIP can mediate alpha-synuclein degradation by two discrete mechanisms that can be dissected using deletion mutants; the tetratricopeptide repeat domain is critical for proteasomal degradation, whereas the U-box domain is sufficient to direct alpha-synuclein toward the lysosomal degradation pathway. Furthermore, alpha-synuclein, synphilin-1, and Hsp70 all coimmunoprecipitate with CHIP, raising the possibility of a direct alpha-synuclein-CHIP interaction. The fact that the tetratricopeptide repeat domain is required for the effects of CHIP on alpha-synuclein inclusion morphology, number of inclusions, and proteasomal degradation as well as the direct interaction of CHIP with Hsp70 implicates a cooperation of CHIP and Hsp70 in these processes. Taken together, these data suggest that CHIP acts a molecular switch between proteasomal and lysosomal degradation pathways.

Cell Line, Tumor↗
PubMed2005

In vivo evidence of CHIP up-regulation attenuating tau aggregation.

The carboxyl terminus of heat-shock cognate (Hsc)70-interacting protein (CHIP) is a ubiquitin E3 ligase that can collaborate with molecular chaperones to facilitate protein folding and prevent protein aggregation. Previous studies showed that, together with heat-shock protein (Hsp)70, CHIP can regulate tau ubiquitination and degradation in a cell culture system. Ubiquitinated tau is one component in neurofibrillary tangles (NFTs), which are a major histopathological feature of Alzheimer's disease (AD). However, the precise sequence of events leading to NFT formation and the mechanisms involved remain unclear. To confirm CHIP's role in suppressing NFT formation in vivo, we performed a quantitative analysis of CHIP in human and mouse brains. We found increased levels of CHIP and Hsp70 in AD compared with normal controls. CHIP levels in both AD and controls corresponded directly to Hsp90 levels, but not to Hsp70 or Hsc70 levels. In AD samples, CHIP was inversely proportional to sarkosyl-insoluble tau accumulation. In a JNPL3 mouse brain tauopathy model, CHIP was widely distributed but weakly expressed in spinal cord, which was the most prominent region for tau inclusions and neuronal loss. Protein levels of CHIP in cerebellar regions of JNPL3 mice were significantly higher than in non-transgenic littermates. Human tau was more highly expressed in this region of mouse brains, but only moderate levels of sarkosyl-insoluble tau were detected. This was confirmed when increased insoluble tau accumulation was found in mice lacking CHIP. These findings suggest that increases in CHIP may protect against NFT formation in the early stages of AD. If confirmed, this would indicate that the quality-control machinery in a neuron might play an important role in retarding the pathogenesis of tauopathies.

Aged↗
PubMed1999

Identification of CHIP, a novel tetratricopeptide repeat-containing protein that interacts with heat shock proteins and negatively regulates chaperone functions.

The chaperone function of the mammalian 70-kDa heat shock proteins Hsc70 and Hsp70 is modulated by physical interactions with four previously identified chaperone cofactors: Hsp40, BAG-1, the Hsc70-interacting protein Hip, and the Hsc70-Hsp90-organizing protein Hop. Hip and Hop interact with Hsc70 via a tetratricopeptide repeat domain. In a search for additional tetratricopeptide repeat-containing proteins, we have identified a novel 35-kDa cytoplasmic protein, carboxyl terminus of Hsc70-interacting protein (CHIP). CHIP is highly expressed in adult striated muscle in vivo and is expressed broadly in vitro in tissue culture. Hsc70 and Hsp70 were identified as potential interaction partners for this protein in a yeast two-hybrid screen. In vitro binding assays demonstrated direct interactions between CHIP and both Hsc70 and Hsp70, and complexes containing CHIP and Hsc70 were identified in immunoprecipitates of human skeletal muscle cells in vivo. Using glutathione S-transferase fusions, we found that CHIP interacted with the carboxy-terminal residues 540 to 650 of Hsc70, whereas Hsc70 interacted with the amino-terminal residues 1 to 197 (containing the tetratricopeptide domain and an adjacent charged domain) of CHIP. Recombinant CHIP inhibited Hsp40-stimulated ATPase activity of Hsc70 and Hsp70, suggesting that CHIP blocks the forward reaction of the Hsc70-Hsp70 substrate-binding cycle. Consistent with this observation, both luciferase refolding and substrate binding in the presence of Hsp40 and Hsp70 were inhibited by CHIP. Taken together, these results indicate that CHIP decreases net ATPase activity and reduces chaperone efficiency, and they implicate CHIP in the negative regulation of the forward reaction of the Hsc70-Hsp70 substrate-binding cycle.

Adenosine Triphosphatases↗
PubMed2003

Overexpression of the cochaperone CHIP enhances Hsp70-dependent folding activity in mammalian cells.

CHIP is a cochaperone of Hsp70 that inhibits Hsp70-dependent refolding in vitro. However, the effect of altered expression of CHIP on the fate of unfolded proteins in mammalian cells has not been determined. Surprisingly, we found that overexpression of CHIP in fibroblasts increased the refolding of proteins after thermal denaturation. This effect was insensitive to geldanamycin, an Hsp90 inhibitor, and required the tetratricopeptide repeat motifs but not the U-box domain of CHIP. Inhibition of Hsp70 chaperone activity abolished the effects of CHIP on protein folding, indicating that the CHIP-mediated events were Hsp70 dependent. Hsp40 competitively inhibited the CHIP-dependent refolding, which is consistent with in vitro data indicating that these cofactors act on Hsp70 in the ATP-bound state and have opposing effects on Hsp70 ATPase activity. Consistent with these observations, CHIP overexpression did not alter protein folding in the setting of ATP depletion, when Hsp70 is in the ADP-bound state. Concomitant with its effects on refolding heat-denatured substrates, CHIP increased the fraction of nascent chains coimmunoprecipitating with Hsc70, but only when sufficient ATP was present to allow Hsp70 to cycle rapidly. Our data suggest that, consistent with in vitro studies, CHIP attenuates the Hsp70 cycle in living cells. The impact of this effect on the fate of unfolded proteins in cells, however, is different from what might be expected from the in vitro data. Rather than resulting in inhibited refolding, CHIP increases the folding capacity of Hsp70 in eukaryotic cells.

Adenosine Triphosphate↗
PubMed2004

CHIP mediates degradation of Smad proteins and potentially regulates Smad-induced transcription.

Transforming growth factor beta (TGF-beta)/bone morphogenetic protein (BMP) family ligands interact with specific membrane receptor complexes that have serine/threonine kinase activities. The receptor phosphorylation and activation induced by the ligands leads to phosphorylation of the Smad proteins, which translocate to the nucleus, controlling gene expression. Thus, regulation of Smad proteins is a key step in TGF-beta/BMP-induced signal transduction. Here we report a novel mechanism of the regulation of SMAD-mediated signaling, by which the Smad1 protein level is controlled through expression of the CHIP protein. CHIP is a U-box-dependent E3 ubiquitin ligase, previously identified as a cochaperon protein. However, we have isolated CHIP as a Smad-interacting protein in a yeast two-hybrid screen using Smad1 as bait. Furthermore we have shown CHIP-Smad interaction using the (35)S-labeled CHIP protein, which can interact with glutathione S-transferase (GST)-Smad1 and GST-Smad4 in an in vitro protein-binding assay. The CHIP-Smad interaction has been confirmed in vivo in mammalian cells through coimmunoprecipitation. Interestingly, we demonstrate that the coexpression of Smad1 and Smad4 with the CHIP protein results in the degradation of the Smad proteins through a ubiquitin-mediated process. Consistent with the observation that CHIP induces Smad1 degradation, we further show that the expression of CHIP can inhibit the transcriptional activities of the Smad1/Smad4 complex induced by BMP signals. Intriguingly, pBS/U6/CHIPi, which diminishes CHIP expression, significantly enhanced Smad1/Smad4- or BMPRIB(QD)-induced gene transcription. These results suggest that CHIP can interact with the Smad1/Smad4 proteins and block BMP signal transduction through the ubiquitin-mediated degradation of Smad proteins.

Binding Sites↗
PubMed2000

[Application of dielectric cooking in the elaboration of instant maize flour for the preparation of corn and tortilla chips].

This present work reports on the evaluation of a method for producing of instant flour for corn and tortilla chips based on dielectric cooking (DC). The samples prepared with lime showed a smaller variation in color than those without lime. A greater water absorption capacity was found in the samples processed without lime. The values for cohesion and adhesion of the DC masa prepared with lime are within the range of values previously reported for samples of masa prepared with nixtamal and commercial instant maize flours. The color analyses of chips from DC masa showed slight changes in relation to the chips prepared by the traditional process of nixtamalization. The DC tortilla chips (plus lime) with 15 minutes of DC showed similar values of delta E as compared to the control (commercial tortilla chips). The moisture of the DC chips varied in the range from 1.5 to 2.8. The DC tortilla chips with lime absorbed a lower amount of oil than the control sample and the corn chips. In fact, the DC chips absorbed lower amount of oil as compared to the control. The DC tortilla chips with lime and processed for 10 and 15 minutes were crunchy and smooth. Dielectric cooking displays a high potential for corn and tortilla chips with good functional characteristics, giving substantial savings of water, processing time and with higher yields due to the use of the whole grain.

Cooking↗
PubMed1995

A general method to recondition and reuse BIAcore sensor chips fouled with covalently immobilized protein/peptide.

Of significance in the routine use of BIAcore is the cost of the sensor chips. This is particularly evident during the phase of method development of an assay where it is not unusual to expend several chips in a day in attempts to optimize immobilization conditions for a novel peptide or protein. In addition, it is accepted practice to discard a chip once its ligand binding capacity has diminished to an unacceptable level. While the high cost of sensor chips has been addressed to some degree through the recent introduction of research-grade sensor chips, we were interested in assessing the possibility of regenerating or reconditioning sensor chips in order to allow them to be reused. In particular, we concerned ourselves with regenerating sensor chips onto which peptide or protein had been immobilized. Our aim was to develop a general procedure that would allow reuse of such chips but would not decrease ligand immobilization capacity or increase nonspecific ligand adsorption properties. We present a method which employs a combination of enzymatic (Pronase E) and chemical (bromoacetic acid) treatments of used sensor chips. Regeneration requires an overnight incubation of the sensor chip ex situ so that one can continue to perform BIAcore experiments. The data demonstrate that this simple two-step procedure substantially removes immobilized proteins such as IgG, Protein G, an HIV-1 envelope glycoprotein (gp 120) and a neoglycoprotein based on bovine serum albumin, as determined by reflectance measurements and X-ray photoelectron spectroscopy.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2005

Detection of HPV genotypes in cervical lesions by the HPV DNA Chip and sequencing.

OBJECTIVE: A newly introduced HPV detection technique in cervical lesion, the HPV DNA Chip test, contains 24 HPV probes and has the advantage of being able to detect 24 HPV types at once. We performed HPV DNA sequencing and compared the results with that of the HPV DNA Chip for evaluation of the accuracy of the DNA Chip test. METHODS: The HPV DNA sequencing was performed in samples of 282 patients, where specific HPV type had been shown in HPV DNA Chip test. The sixteen cases where multiple HPV types had been found in HPV DNA Chip test were included in 282 cases. The sequencing was also performed in HPV-other type samples of 95 patients, where positive in HPV-PCR, but specific HPV type had not been found. RESULTS: In 257 cases (91.1%) of 282 cases, the HPV types of the HPV DNA sequencing test were in agreement with types of the HPV DNA Chip. In 16 cases (5.7%), the sequencing types were different from the types of HPV DNA Chip. But, in 9 of 16 cases, types in HPV DNA sequencing were absent types in HPV DNA Chip test. The interpretation of HPV DNA sequencing was impossible in nine cases (3.2%). The HPV DNA sequencing test of 95 cases of HPV-other type showed that the sequencing types from 94 cases (98.9%) were absent types in HPV DNA Chip test. In sequencing test of HPV-other type, HPV-81 (20.0%), HPV-62 (14.7%), HPV-84 (13.7%), and HPV-61 (13.7%) were frequently detected. CONCLUSION: HPV DNA Chip is an accurate method for detecting the 24 HPV genotypes.

Base Sequence↗
PubMed1993

Distribution of the aquaporin CHIP in secretory and resorptive epithelia and capillary endothelia.

The existence of water-selective channels has been postulated to explain the high water permeability of erythrocytes and certain epithelial cells. The aquaporin CHIP (channel-forming integral membrane protein of 28 kDa), a molecular water channel, is abundant in erythrocytes and water-permeable segments of the nephron. To determine whether CHIP may mediate transmembrane water movement in other water-permeable epithelia, membranes of multiple organs were studied by immunoblotting, immunohistochemistry, and immunoelectron microscopy using affinity-purified anti-CHIP IgG. The apical membrane of the choroid plexus epithelium was densely stained, implying a role for CHIP in the secretion of cerebrospinal fluid. In the eye, CHIP was abundant in apical and basolateral domains of ciliary epithelium, the site of aqueous humor secretion, and also in lens epithelium and corneal endothelium. CHIP was detected in membranes of hepatic bile ducts and water-resorptive epithelium of gall bladder, suggesting a role in bile secretion and concentration. CHIP was not detected in glandular epithelium of mammary, salivary, or lacrimal glands, suggesting the existence of other water-channel isoforms. CHIP was also not detected within the epithelium of the gastrointestinal mucosa. CHIP was abundant in membranes of intestinal lacteals and continuous capillaries in diverse tissues, including cardiac and skeletal muscle, thus providing a molecular explanation for the known water permeability of certain lymphatics and capillary beds. These studies underscore the hypothesis that CHIP plays a major role in transcellular water movement throughout the body.

Animals↗
PubMed2006

CHIP protects from the neurotoxicity of expanded and wild-type ataxin-1 and promotes their ubiquitination and degradation.

CHIP (C terminus of Hsc-70 interacting protein) is an E3 ligase that links the protein folding machinery with the ubiquitin-proteasome system and has been implicated in disorders characterized by protein misfolding and aggregation. Here we investigate the role of CHIP in protecting from ataxin-1-induced neurodegeneration. Ataxin-1 is a polyglutamine protein whose expansion causes spinocerebellar ataxia type-1 (SCA1) and triggers the formation of nuclear inclusions (NIs). We find that CHIP and ataxin-1 proteins directly interact and co-localize in NIs both in cell culture and SCA1 postmortem neurons. CHIP promotes ubiquitination of expanded ataxin-1 both in vitro and in cell culture. The Hsp70 chaperone increases CHIP-mediated ubiquitination of ataxin-1 in vitro, and the tetratricopeptide repeat domain, which mediates CHIP interactions with chaperones, is required for ataxin-1 ubitiquination in cell culture. Interestingly, CHIP also interacts with and ubiquitinates unexpanded ataxin-1. Overexpression of CHIP in a Drosophila model of SCA1 decreases the protein steady-state levels of both expanded and unexpanded ataxin-1 and suppresses their toxicity. Finally we investigate the ability of CHIP to protect against toxicity caused by expanded polyglutamine tracts in different protein contexts. We find that CHIP is not effective in suppressing the toxicity caused by a bare 127Q tract with only a short hemagglutinin tag, but it is very efficient in suppressing toxicity caused by a 128Q tract in the context of an N-terminal huntingtin backbone. These data underscore the importance of the protein framework for modulating the effects of polyglutamine-induced neurodegeneration.

Animals↗
PubMed1997

Chip, a widely expressed chromosomal protein required for segmentation and activity of a remote wing margin enhancer in Drosophila.

The mechanisms allowing remote enhancers to regulate promoters several kilobase pairs away are unknown but are blocked by the Drosophila suppressor of Hairy-wing protein (Suhw) that binds to gypsy retrovirus insertions between enhancers and promoters. Suhw bound to a gypsy insertion in the cut gene also appears to act interchromosomally to antagonize enhancer-promoter interactions on the homologous chromosome when activity of the Chip gene is reduced. This implicates Chip in enhancer-promoter communication. We cloned Chip and find that it encodes a homolog of the recently discovered mouse Nli/Ldb1/Clim-2 and Xenopus Xldb1 proteins that bind nuclear LIM domain proteins. Chip protein interacts with the LIM domains in the Apterous homeodomain protein, and Chip interacts genetically with apterous, showing that these interactions are important for Apterous function in vivo. Importantly, Chip also appears to have broad functions beyond interactions with LIM domain proteins. Chip is present in all nuclei examined and at numerous sites along the salivary gland polytene chromosomes. Embryos without Chip activity lack segments and show abnormal gap and pair-rule gene expression, although no LIM domain proteins are known to regulate segmentation. We conclude that Chip is a ubiquitous chromosomal factor required for normal expression of diverse genes at many stages of development. We suggest that Chip cooperates with different LIM domain proteins and other factors to structurally support remote enhancer-promoter interactions.

Amino Acid Sequence↗
PubMed2002

Fat content of chips, quality of frying fat and deep-frying practices in New Zealand fast food outlets.

OBJECTIVES: To collect baseline data on the fat content of hot chips, quality (degradation) of cooking fat, deep-frying practices and related attitudes in fast food outlets in New Zealand. To identify the key determinants of the fat content of chips and quality of cooking fat. METHODS: A nationally representative sample of fast food outlets (n=150, response rate 80%) was surveyed between September 1998 and March 1999. Data collected included a questionnaire, observation of cooking practices and analysis of cooked chips and frying fat. RESULTS: Only 8% of independent operators had formal training in deep frying practices compared with 93% of chain operators. There was a wide range of fat content of chips (5%-20%, mean 11.5%). The use of thinner chips, crinkle cut chips and lower fryer fat temperature were associated with higher chip fat content. Eighty-nine per cent of chain outlets used 6-10 mm chips compared with 83% of independent outlets that used chips > or = 12 mm. A wide range of frying temperatures was recorded (136-233 degrees C) with 58% of outlets frying outside the reference range (175-190 degrees C). As indices of fat degradation, fat acid and polar compound values above the recommended levels occurred in 54% and 5% of outlets respectively. Operators seemed willing to learn more about best practice techniques, with lack of knowledge being the main barrier to change. CONCLUSIONS AND IMPLICATIONS: Deep frying practices could be improved through operator training and certification options. Even a small decrease in the mean fat content of chips would reduce the obesogenic impact of this popular food.

Cooking↗
PubMed2004

Notch-induced E2A degradation requires CHIP and Hsc70 as novel facilitators of ubiquitination.

E2A transcription factors, E12 and E47, are important regulators of lymphocyte development. Notch signaling pathways have been shown to regulate E2A function by accelerating the degradation of E2A proteins through a mitogen-activated protein kinase-dependent and ubiquitin-mediated pathway. To further understand the mechanism underlying E2A ubiquitination and degradation, we conducted a yeast two-hybrid screen and identified the carboxyl terminus of Hsc70-interacting protein (CHIP) as an E47 binding protein. Here, we show that CHIP associates with E2A proteins in vivo and that overexpression of CHIP induces E47 degradation in a phosphorylation-dependent manner. Conversely, knocking down CHIP with small interfering RNA alleviates Notch-induced E47 degradation. CHIP binds E47 through the E protein homology domains 2 and 3 (EHD2 and EHD3). This interaction between CHIP and E47 is independent of the U-box domain with E3 ubiquitin ligase activity but requires the chaperone binding tetratricopeptide repeats domain. The ability of CHIP to induce E47 ubiquitination and degradation correlates with its ability to bind E47. We propose that CHIP, together with its partner Hsc70, forms a preubiquitination complex (PUC) with E47 and Skp2, thus facilitating the interaction between E47 and Skp2. CHIP also associates with Cul1, which introduces PUC to the SCF E3 ligase complex, responsible for E47 ubiquitination. Therefore, CHIP plays a crucial role in the ubiquitination and degradation of E2A proteins.

Amino Acid Sequence↗
PubMed1993

Aquaporin CHIP: the archetypal molecular water channel.

Despite longstanding interest by nephrologists and physiologists, the molecular identities of membrane water channels remained elusive until recognition of CHIP, a 28-kDa channel-forming integral membrane protein from human red blood cells originally referred to as "CHIP28." CHIP functions as an osmotically driven, water-selective pore; 1) expression of CHIP conferred Xenopus oocytes with markedly increased osmotic water permeability but did not allow transmembrane passage of ions or other small molecules; 2) reconstitution of highly purified CHIP into proteoliposomes permitted determination of the unit water permeability, i.e., 3.9 x 10(9) water molecules.channel subunit-1 x s-1. Although CHIP exists as a homotetramer in the native red blood cell membrane, site-directed mutagenesis studies suggested that each subunit contains an individually functional pore that may be reversibly occluded by mercurial inhibitors reacting with cysteine-189. CHIP is a major component of both apical and basolateral membranes of water-permeable segments of the nephron, where it facilitates transcellular water flow during reabsorption of glomerular filtrate. CHIP is also abundant in certain other absorptive or secretory epithelia, including choroid plexus, ciliary body of the eye, hepatobiliary ductules, gall bladder, and capillary endothelia. Distinct patterns of CHIP expression occur at these sites during fetal development and maturity. Similar proteins from other mammalian tissues and plants were later shown to transport water, and the group is now referred to as the "aquaporins." Recognition of CHIP has provided molecular insight into the biological phenomenon of osmotic water movement, and it is hoped that pharmacological modulation of CHIP function may provide novel treatments of renal failure and other clinical problems.

Amino Acid Sequence↗
PubMed1995

Quantification of Aquaporin-CHIP water channel protein in microdissected renal tubules by fluorescence-based ELISA.

Several transporters have been localized along the nephron by physiological methods or immunocytochemistry. However, the actual abundance of these molecules has not been established. To accomplish this goal, we have developed a fluorescence-based ELISA method and have used it to quantitate Aquaporin-CHIP (AQP-CHIP) water channel protein in rat kidney tubules. Microdissected tubules (2 mm/sample, permeabilized with 0.5% Triton X-100) or purified AQP-CHIP standards (0-200 fmol) were utilized in a fluorescence ELISA protocol after covalent immobilization on epoxy-activated Sepharose beads. The lower limit of detection was 2.4 fmol of AQP-CHIP. Preabsorption with excess purified AQP-CHIP or use of nonimmune serum eliminated the signal. In proximal segments, the measured AQP-CHIP was linearly related to tubule length (1-10 mm). The measured AQP-CHIP was (mean +/- SE, fmol/mm): S-1 proximal, 10.8 +/- 2.1; S-2, 10.0 +/- 2.3; S-3, 21.3 +/- 3.1; type 1 thin descending limb (DTL), 12.9 +/- 4.6; type 2 DTL, 86.5 +/- 19.5; type 3 DTL, 43.0 +/- 11.2. In thin ascending limbs, thick ascending limbs, distal convoluted tubules, connecting tubules, and collecting ducts, the AQP-CHIP signal was indistinguishable from zero. Based on the unit water conductance of single CHIP molecules, our calculations show that the content of AQP-CHIP is sufficient to explain water permeability measured in isolated proximal tubules and DTL segments.

Animals↗