Статті в журналах: "Protei chip"

1

Tsujibo, Hiroshi, Hideyuki Orikoshi, Nao Baba, Masahiro Miyahara, Katsushiro Miyamoto, Masahide Yasuda, and Yoshihiko Inamori. "Identification and Characterization of the Gene Cluster Involved in Chitin Degradation in a Marine Bacterium, Alteromonas sp. Strain O-7." Applied and Environmental Microbiology 68, no. 1 (January 2002): 263–70. http://dx.doi.org/10.1128/aem.68.1.263-270.2002.

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ABSTRACT Alteromonas sp. strain O-7 secretes chitinase A (ChiA), chitinase B (ChiB), and chitinase C (ChiC) in the presence of chitin. A gene cluster involved in the chitinolytic system of the strain was cloned and sequenced upstream of and including the chiA gene. The gene cluster consisted of three different open reading frames organized in the order chiD, cbp1, and chiA. The chiD, cbp1, and chiA genes were closely linked and transcribed in the same direction. Sequence analysis indicated that Cbp1 (475 amino acids) was a chitin-binding protein composed of two discrete functional regions. ChiD (1,037 amino acids) showed sequence similarity to bacterial chitinases classified into family 18 of glycosyl hydrolases. The cbp1 and chiD genes were expressed in Escherichia coli, and the recombinant proteins were purified to homogeneity. The highest binding activities of Cbp1 and ChiD were observed when α-chitin was used as a substrate. Cbp1 and ChiD possessed a chitin-binding domain (ChtBD) belonging to ChtBD type 3. ChiD rapidly hydrolyzed chitin oligosaccharides in sizes from trimers to hexamers, but not chitin. However, after prolonged incubation with large amounts of ChiD, the enzyme produced a small amount of (GlcNAc)2 from chitin. The optimum temperature and pH of ChiD were 50°C and 7.0, respectively.

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2

Orikoshi, Hideyuki, Nao Baba, Shigenari Nakayama, Hiroshi Kashu, Katsushiro Miyamoto, Masahide Yasuda, Yoshihiko Inamori, and Hiroshi Tsujibo. "Molecular Analysis of the Gene Encoding a Novel Cold-Adapted Chitinase (ChiB) from a Marine Bacterium, Alteromonas sp. Strain O-7." Journal of Bacteriology 185, no. 4 (February 15, 2003): 1153–60. http://dx.doi.org/10.1128/jb.185.4.1153-1160.2003.

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ABSTRACT The chitinase B (ChiB) secreted by Alteromonas sp. strain O-7 was purified, and the corresponding gene (chiB) was cloned and sequenced. The open reading frame of the chiB gene encodes a protein of 850 amino acids with a calculated molecular mass of 90,223 Da. ChiB is a modular enzyme consisting of two reiterated domains and a catalytic domain belonging to chitinase family 18. The reiterated domains are composed of chitin-binding domain (ChtBD) type 3 and two fibronectin type III (Fn3)-like domains. Expression plasmids coding for ChiB or deletion derivatives thereof were constructed in Escherichia coli. Deletion analysis showed that the ChtBD of ChiB plays an important role in efficient hydrolysis of insoluble chitin. The optimum pH and temperature of ChiB were 6.0 and 30°C, respectively. The enzyme showed relatively high catalysis, even at low temperatures close to 0°C, and remarkable thermal lability compared to ChiA and ChiC, which are the mesophilic chitinases of the same strain. The k cat/K m value for the ChiB reaction at 10°C was about 4.7 times higher than that of ChiC. These results suggest that ChiB is a cold-adapted enzyme. The RNA transcript of chiB was induced by 1% GlcNAc, and along with a rise in temperature, the RNA transcript showed a tendency to decrease. Thus, among the ChiA, ChiB, and ChiC chitinases, production of ChiB may be advantageous for the strain, allowing it to easily acquire nutrients from chitin and to survive in cold environments.

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Kim, Yeon-Ki, Zhi-Mei Liu, Daoxin Li, and Pappachan E. Kolattukudy. "Two Novel Genes Induced by Hard-Surface Contact ofColletotrichum gloeosporioides Conidia." Journal of Bacteriology 182, no. 17 (September 1, 2000): 4688–95. http://dx.doi.org/10.1128/jb.182.17.4688-4695.2000.

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ABSTRACT Germinating conidia of many phytopathogenic fungi must differentiate into an infection structure called the appressorium in order to penetrate into their hosts. This differentiation is known to require contact with a hard surface. However, the molecular basis for this requirement is not known. Induction of this differentiation in the avocado pathogen, Colletotrichum gloeosporioides, by chemical signals such as the host's surface wax or the fruit-ripening hormone, ethylene, requires contact of the conidia with a hard surface for about 2 h. To study molecular events triggered by hard-surface contact, we isolated several genes expressed during the early stage of hard-surface treatment by a differential-display method. The genes that encode Colletotrichum hard-surface induced proteins are designated chip genes. In this study, we report the characterization of CHIP2 and CHIP3 genes that would encode proteins with molecular masses of 65 and 64 kDa, respectively, that have no homology to any known proteins. TheCHIP2 product would contain a putative nuclear localization signal, a leucine zipper motif, and a heptad repeat region which might dimerize into coiled-coil structure. The CHIP3 product would be a nine-transmembrane-domain-containing protein. RNA blots showed that CHIP2 and CHIP3 are induced by a 2-h hard-surface contact. However, disruption of these genes did not affect the appressorium-forming ability and did not cause a significant decrease in virulence on avocado or tomato fruits suggesting thatC. gloeosporioides might have genes functionally redundant to CHIP2 and CHIP3 or that these genes induced by hard-surface contact control processes not directly involved in pathogenesis.

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4

Hamilton, Jaeger J., Victoria L. Marlow, Richard A. Owen, Marília de Assis Alcoforado Costa, Manman Guo, Grant Buchanan, Govind Chandra, et al. "A holin and an endopeptidase are essential for chitinolytic protein secretion in Serratia marcescens." Journal of Cell Biology 207, no. 5 (December 8, 2014): 615–26. http://dx.doi.org/10.1083/jcb.201404127.

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Pathogenic bacteria adapt to their environment and manipulate the biochemistry of hosts by secretion of effector molecules. Serratia marcescens is an opportunistic pathogen associated with healthcare-acquired infections and is a prolific secretor of proteins, including three chitinases (ChiA, ChiB, and ChiC) and a chitin binding protein (Cbp21). In this work, genetic, biochemical, and proteomic approaches identified genes that were required for secretion of all three chitinases and Cbp21. A genetic screen identified a holin-like protein (ChiW) and a putative l-alanyl-d-glutamate endopeptidase (ChiX), and subsequent biochemical analyses established that both were required for nonlytic secretion of the entire chitinolytic machinery, with chitinase secretion being blocked at a late stage in the mutants. In addition, live-cell imaging experiments demonstrated bimodal and coordinated expression of chiX and chiA and revealed that cells expressing chiA remained viable. It is proposed that ChiW and ChiX operate in tandem as components of a protein secretion system used by gram-negative bacteria.

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5

Coorey, Ranil, Alexandra Grant, and Vijay Jayasena. "Effects of Chia Flour Incorporation on the Nutritive Quality and Consumer Acceptance of Chips." Journal of Food Research 1, no. 4 (October 25, 2012): 85. http://dx.doi.org/10.5539/jfr.v1n4p85.

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<p>Gluten free, antioxidant, calcium and dietary fibre rich, chia is known to contain the highest level of omega-3 available in any cultivated plant source. The objective of this research was to develop a high protein, high dietary fibre, gluten free and omega-3 fatty acid rich chips. Four different levels of whole chia flour (5%, 10%, 12%, and 15%) were incorporated to produce chia chip. There were no significant differences in appearance, colour, flavour and overall liking between a commercial chip sample and the 5% chia chips. The chemical analysis indicated that all four trial chips are excellent sources of omega-3 and the baking process has a limited impact on their nutritional profile. For optimal consumer acceptance and nutritional benefits, the incorporation of 5% chia is recommended. With limited chia based food products currently available, a chia chip would be a well-accepted and healthy alternative to the common unhealthy chips.</p>

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6

Folders, Jindra, Jon Algra, Marc S. Roelofs, Leendert C. van Loon, Jan Tommassen, and Wilbert Bitter. "Characterization of Pseudomonas aeruginosa Chitinase, a Gradually Secreted Protein." Journal of Bacteriology 183, no. 24 (December 15, 2001): 7044–52. http://dx.doi.org/10.1128/jb.183.24.7044-7052.2001.

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ABSTRACT The gram-negative bacterium Pseudomonas aeruginosasecretes many proteins into its extracellular environment via the type I, II, and III secretion systems. In this study, a gene,chiC, coding for an extracellular chitinolytic enzyme, was identified. The chiC gene encodes a polypeptide of 483 amino acid residues, without a typical N-terminal signal sequence. Nevertheless, an N-terminal segment of 11 residues was found to be cleaved off in the secreted protein. The protein shows sequence similarity to the secreted chitinases ChiC of Serratia marcescens, ChiA of Vibrio harveyi, and ChiD ofBacillus circulans and consists of an activity domain and a chitin-binding domain, which are separated by a fibronectin type III domain. ChiC was able to bind and degrade colloidal chitin and was active on the artificial substrates carboxymethyl-chitin-Remazol Brilliant Violet andp-nitrophenyl-β-d-N,N′,N"-triacetylchitotriose, but not onp-nitrophenyl-β-d-N-acetylglucosamine, indicating that it is an endochitinase. Expression of thechiC gene appears to be regulated by the quorum-sensing system of P. aeruginosa, since this gene was not expressed in a lasIR vsmI mutant. After overnight growth, the majority of the ChiC produced was found intracellularly, whereas only small amounts were detected in the culture medium. However, after several days, the cellular pool of ChiC was largely depleted, and the protein was found in the culture medium. This release could not be ascribed to cell lysis. Since ChiC did not appear to be secreted via any of the known secretion systems, a novel secretion pathway seems to be involved.

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7

Tsujibo, Hiroshi, Takahiro Kubota, Mitsugu Yamamoto, Katsushiro Miyamoto, and Yoshihiko Inamori. "Characterization of Chitinase Genes from an Alkaliphilic Actinomycete, Nocardiopsis prasina OPC-131." Applied and Environmental Microbiology 69, no. 2 (February 2003): 894–900. http://dx.doi.org/10.1128/aem.69.2.894-900.2003.

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ABSTRACT An alkaliphilic actinomycete, Nocardiopsis prasina OPC-131, secretes chitinases, ChiA, ChiB, and ChiBΔ, in the presence of chitin. The genes encoding ChiA and ChiB were cloned and sequenced. The open reading frame (ORF) of chiA encoded a protein of 336 amino acids with a calculated molecular mass of 35,257 Da. ChiA consisted of only a catalytic domain and showed a significant homology with family 18 chitinases. The chiB ORF encoded a protein of 296 amino acids with a calculated molecular mass of 31,500 Da. ChiB is a modular enzyme consisting of a chitin-binding domain type 3 (ChtBD type 3) and a catalytic domain. The catalytic domain of ChiB showed significant similarity to Streptomyces family 19 chitinases. ChiBΔ was the truncated form of ChiB lacking ChtBD type 3. Expression plasmids coding for ChiA, ChiB, and ChiBΔ were constructed to investigate the biochemical properties of these recombinant proteins. These enzymes showed pHs and temperature optima similar to those of native enzymes. ChiB showed more efficient hydrolysis of chitin and stronger antifungal activity than ChiBΔ, indicating that the ChtBD type 3 of ChiB plays an important role in the efficient hydrolysis of chitin and in antifungal activity. Furthermore, the finding of family 19 chitinase in N. prasina OPC-131 suggests that family 19 chitinases are distributed widely in actinomycetes other than the genus Streptomyces.

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8

Lee, Yoonsuk, Dong-Ku Kang, Soo-Ik Chang, Moon Hi Han, and In-Cheol Kang. "High-Throughput Screening of Novel Peptide Inhibitors of an Integrin Receptor from the Hexapeptide Library by Using a Protein Microarray Chip." Journal of Biomolecular Screening 9, no. 8 (December 2004): 687–94. http://dx.doi.org/10.1177/1087057104268125.

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Protein microarray is an emerging technology that makes high-throughput analysis possible for protein-protein interactions and analysis of proteome and biomarkers in parallel. The authors investigated the application of a novel protein microarray chip, Proteo Chip, in new drug discovery. Integrin αvβ3 microarray immobilized on the Proteo Chip was employed to screen new active peptides against the integrin from multiple hexapeptide sublibraries of a positional scanning synthetic peptide combinatorial library (PS-SPCL). The integrin αvβ3-vitronectin interaction was successfully demonstrated on the integrin microarray in a dose-dependent manner andwas inhibited not only by the syntheticRGDpeptide but also by various integrin antagonists on the integrin microarray chip. Novel peptide ligands with high affinity to the integrin were also identified from the peptide libraries with this chip-based screening system by a competitive inhibition assay in a simultaneous and highthroughput fashion. The authors have confirmed antiangiogenic functions of the novel peptides thus screened through an in vitro and in vivo angiogenesis assay. These results provide evidence that the Proteo Chip is a promising tool for highthroughput screening of lead molecules in new drug development.

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9

Zhu, Heng, and Michael Snyder. "Protein chip technology." Current Opinion in Chemical Biology 7, no. 1 (February 2003): 55–63. http://dx.doi.org/10.1016/s1367-5931(02)00005-4.

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10

Howard, Michael B., Nathan A. Ekborg, Larry E. Taylor, Ronald M. Weiner, and Steven W. Hutcheson. "Genomic Analysis and Initial Characterization of the Chitinolytic System of Microbulbifer degradans Strain 2-40." Journal of Bacteriology 185, no. 11 (June 1, 2003): 3352–60. http://dx.doi.org/10.1128/jb.185.11.3352-3360.2003.

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ABSTRACT The marine bacterium Microbulbifer degradans strain 2-40 produces at least 10 enzyme systems for degrading insoluble complex polysaccharides (ICP). The draft sequence of the 2-40 genome allowed a genome-wide analysis of the chitinolytic system of strain 2-40. The chitinolytic system includes three secreted chitin depolymerases (ChiA, ChiB, and ChiC), a secreted chitin-binding protein (CbpA), periplasmic chitooligosaccharide-modifying enzymes, putative sugar transporters, and a cluster of genes encoding cytoplasmic proteins involved in N-acetyl-d-glucosamine (GlcNAc) metabolism. Each chitin depolymerase was detected in culture supernatants of chitin-grown strain 2-40 and was active against chitin and glycol chitin. The chitin depolymerases also had a specific pattern of activity toward the chitin analogs 4-methylumbelliferyl-β-d-N,N′-diacetylchitobioside (MUF-diNAG) and 4-methylumbelliferyl-β-d-N,N′,N"-triacetylchitotrioside (MUF-triNAG). The depolymerases were modular in nature and contained glycosyl hydrolase family 18 domains, chitin-binding domains, and polycystic kidney disease domains. ChiA and ChiB each possessed polyserine linkers of up to 32 consecutive serine residues. In addition, ChiB and CbpA contained glutamic acid-rich domains. At 1,271 amino acids, ChiB is the largest bacterial chitinase reported to date. A chitodextrinase (CdxA) with activity against chitooligosaccharides (degree of polymerization of 5 to 7) was identified. The activities of two apparent periplasmic (HexA and HexB) N-acetyl-β-d-glucosaminidases and one cytoplasmic (HexC) N-acetyl-β-d-glucosaminidase were demonstrated. Genes involved in GlcNAc metabolism, similar to those of the Escherichia coli K-12 NAG utilization operon, were identified. NagA from strain 2-40, a GlcNAc deacetylase, was shown to complement a nagA mutation in E. coli K-12. Except for the GlcNAc utilization cluster, genes for all other components of the chitinolytic system were dispersed throughout the genome. Further examination of this system may provide additional insight into the mechanisms by which marine bacteria degrade chitin and provide a basis for future research on the ICP-degrading systems of strain 2-40.

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Timm, Christopher, and Christof M. Niemeyer. "On-Chip Protein Biosynthesis." Angewandte Chemie International Edition 52, no. 10 (January 10, 2013): 2652–54. http://dx.doi.org/10.1002/anie.201208880.

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12

Ma, Tao, Zhenqing Ye, and Liguo Wang. "Genome Wide Approaches to Identify Protein-DNA Interactions." Current Medicinal Chemistry 26, no. 42 (January 8, 2020): 7641–54. http://dx.doi.org/10.2174/0929867325666180530115711.

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Background: Transcription factors are DNA-binding proteins that play key roles in many fundamental biological processes. Unraveling their interactions with DNA is essential to identify their target genes and understand the regulatory network. Genome-wide identification of their binding sites became feasible thanks to recent progress in experimental and computational approaches. ChIP-chip, ChIP-seq, and ChIP-exo are three widely used techniques to demarcate genome-wide transcription factor binding sites. Objective: This review aims to provide an overview of these three techniques including their experiment procedures, computational approaches, and popular analytic tools. Conclusion: ChIP-chip, ChIP-seq, and ChIP-exo have been the major techniques to study genome- wide in vivo protein-DNA interaction. Due to the rapid development of next-generation sequencing technology, array-based ChIP-chip is deprecated and ChIP-seq has become the most widely used technique to identify transcription factor binding sites in genome-wide. The newly developed ChIP-exo further improves the spatial resolution to single nucleotide. Numerous tools have been developed to analyze ChIP-chip, ChIP-seq and ChIP-exo data. However, different programs may employ different mechanisms or underlying algorithms thus each will inherently include its own set of statistical assumption and bias. So choosing the most appropriate analytic program for a given experiment needs careful considerations. Moreover, most programs only have command line interface so their installation and usage will require basic computation expertise in Unix/Linux.

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Zhang, Henry B., Minji Kim, Jeffrey H. Chuang, and Yijun Ruan. "pyBedGraph: a python package for fast operations on 1D genomic signal tracks." Bioinformatics 36, no. 10 (February 11, 2020): 3234–35. http://dx.doi.org/10.1093/bioinformatics/btaa061.

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Abstract Motivation Modern genomic research is driven by next-generation sequencing experiments such as ChIP-seq and ChIA-PET that generate coverage files for transcription factor binding, as well as DHS and ATAC-seq that yield coverage files for chromatin accessibility. Such files are in a bedGraph text format or a bigWig binary format. Obtaining summary statistics in a given region is a fundamental task in analyzing protein binding intensity or chromatin accessibility. However, the existing Python package for operating on coverage files is not optimized for speed. Results We developed pyBedGraph, a Python package to quickly obtain summary statistics for a given interval in a bedGraph or a bigWig file. When tested on 12 ChIP-seq, ATAC-seq, RNA-seq and ChIA-PET datasets, pyBedGraph is on average 260 times faster than the existing program pyBigWig. On average, pyBedGraph can look up the exact mean signal of 1 million regions in ∼0.26 s and can compute their approximate means in <0.12 s on a conventional laptop. Availability and implementation pyBedGraph is publicly available at https://github.com/TheJacksonLaboratory/pyBedGraph under the MIT license. Supplementary information Supplementary data are available at Bioinformatics online.

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ESPEJO, Alexsandra, Jocelyn CÔTÉ, Andrzej BEDNAREK, Stephane RICHARD, and Mark T. BEDFORD. "A protein-domain microarray identifies novel protein–protein interactions." Biochemical Journal 367, no. 3 (November 1, 2002): 697–702. http://dx.doi.org/10.1042/bj20020860.

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Protein domains mediate protein—protein interactions through binding to short peptide motifs in their corresponding ligands. These peptide recognition modules are critical for the assembly of multiprotein complexes. We have arrayed glutathione S-transferase (GST) fusion proteins, with a focus on protein interaction domains, on to nitrocellulose-coated glass slides to generate a protein-domain chip. Arrayed protein-interacting modules included WW (a domain with two conserved tryptophans), SH3 (Src homology 3), SH2, 14.3.3, FHA (forkhead-associated), PDZ (a domain originally identified in PSD-95, DLG and ZO-1 proteins), PH (pleckstrin homology) and FF (a domain with two conserved phenylalanines) domains. Here we demonstrate, using peptides, that the arrayed domains retain their binding integrity. Furthermore, we show that the protein-domain chip can ‘fish’ proteins out of a total cell lysate; these domain-bound proteins can then be detected on the chip with a specific antibody, thus producing an interaction map for a cellular protein of interest. Using this approach we have confirmed the domain-binding profile of the signalling molecule Sam68 (Src-associated during mitosis 68), and have identified a new binding profile for the core small nuclear ribonucleoprotein SmB′. This protein-domain chip not only identifies potential binding partners for proteins, but also promises to recognize qualitative differences in protein ligands (caused by post-translational modification), thus getting at the heart of signal transduction pathways.

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15

Wang, Le, Yi-Tong Liu, Rui Hao, Lei Chen, Zhijie Chang, Hong-Rui Wang, Zhi-Xin Wang, and Jia-Wei Wu. "Molecular Mechanism of the Negative Regulation of Smad1/5 Protein by Carboxyl Terminus of Hsc70-interacting Protein (CHIP)." Journal of Biological Chemistry 286, no. 18 (March 16, 2011): 15883–94. http://dx.doi.org/10.1074/jbc.m110.201814.

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The transforming growth factor-β (TGF-β) superfamily of ligands signals along two intracellular pathways, Smad2/3-mediated TGF-β/activin pathway and Smad1/5/8-mediated bone morphogenetic protein pathway. The C terminus of Hsc70-interacting protein (CHIP) serves as an E3 ubiquitin ligase to mediate the degradation of Smad proteins and many other signaling proteins. However, the molecular mechanism for CHIP-mediated down-regulation of TGF-β signaling remains unclear. Here we show that the extreme C-terminal sequence of Smad1 plays an indispensable role in its direct association with the tetratricopeptide repeat (TPR) domain of CHIP. Interestingly, Smad1 undergoes CHIP-mediated polyubiquitination in the absence of molecular chaperones, and phosphorylation of the C-terminal SXS motif of Smad1 enhances the interaction and ubiquitination. We also found that CHIP preferentially binds to Smad1/5 and specifically disrupts the core signaling complex of Smad1/5 and Smad4. We determined the crystal structures of CHIP-TPR in complex with the phosphorylated/pseudophosphorylated Smad1 peptides and with an Hsp70/Hsc70 C-terminal peptide. Structural analyses and subsequent biochemical studies revealed that the distinct CHIP binding affinities of Smad1/5 or Smad2/3 result from the nonconservative hydrophobic residues at R-Smad C termini. Unexpectedly, the C-terminal peptides from Smad1 and Hsp70/Hsc70 bind in the same groove of CHIP-TPR, and heat shock proteins compete with Smad1/5 for CHIP interaction and concomitantly suppress, rather than facilitate, CHIP-mediated Smad ubiquitination. Thus, we conclude that CHIP inhibits the signaling activities of Smad1/5 by recruiting Smad1/5 from the functional R-/Co-Smad complex and further promoting the ubiquitination/degradation of Smad1/5 in a chaperone-independent manner.

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Service, Robert F. "Protein Chip Promises Cheaper Diagnostics." Science 322, no. 5909 (December 19, 2008): 1784.2–1785. http://dx.doi.org/10.1126/science.322.5909.1784b.

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Severin, Philip M. D., and Hermann E. Gaub. "DNA-Protein Binding Force Chip." Small 8, no. 21 (August 9, 2012): 3269–73. http://dx.doi.org/10.1002/smll.201201088.

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Ronnebaum, Sarah M., Yaxu Wu, Holly McDonough, and Cam Patterson. "The Ubiquitin Ligase CHIP Prevents SirT6 Degradation through Noncanonical Ubiquitination." Molecular and Cellular Biology 33, no. 22 (September 16, 2013): 4461–72. http://dx.doi.org/10.1128/mcb.00480-13.

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The ubiquitin ligase CHIP (carboxyl terminus of Hsp70-interacting protein) regulates protein quality control, and CHIP deletion accelerates aging and reduces the life span in mice. Here, we reveal a mechanism for CHIP's influence on longevity by demonstrating that CHIP stabilizes the sirtuin family member SirT6, a lysine deacetylase/ADP ribosylase involved in DNA repair, metabolism, and longevity. In CHIP-deficient cells, SirT6 protein half-life is substantially reduced due to increased proteasome-mediated degradation, but CHIP overexpression in these cells increases SirT6 protein expression without affecting SirT6 transcription. CHIP noncanonically ubiquitinates SirT6 at K170, which stabilizes SirT6 and prevents SirT6 canonical ubiquitination by other ubiquitin ligases. In CHIP-depleted cells, SirT6 K170 mutation increases SirT6 half-life and prevents proteasome-mediated degradation. The global decrease in SirT6 expression in the absence of CHIP is associated with decreased SirT6 promoter occupancy, which increases histone acetylation and promotes downstream gene transcription in CHIP-depleted cells. Cells lacking CHIP are hypersensitive to DNA-damaging agents, but DNA repair and cell viability are rescued by enforced expression of SirT6. The discovery of this CHIP-SirT6 interaction represents a novel protein-stabilizing mechanism and defines an intersection between protein quality control and epigenetic regulation to influence pathways that regulate the biology of aging.

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19

Tsujibo, Hiroshi, Hideyuki Orikoshi, Kayoko Shiotani, Miyuki Hayashi, Junko Umeda, Katsushiro Miyamoto, Chiaki Imada, Yoshiro Okami, and Yoshihiko Inamori. "Characterization of Chitinase C from a Marine Bacterium, Alteromonas sp. Strain O-7, and Its Corresponding Gene and Domain Structure." Applied and Environmental Microbiology 64, no. 2 (February 1, 1998): 472–78. http://dx.doi.org/10.1128/aem.64.2.472-478.1998.

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ABSTRACT One of the chitinase genes of Alteromonas sp. strain O-7, the chitinase C-encoding gene (chiC), was cloned, and the nucleotide sequence was determined. An open reading frame coded for a protein of 430 amino acids with a predicted molecular mass of 46,680 Da. Alignment of the deduced amino acid sequence demonstrated that ChiC contained three functional domains, the N-terminal domain, a fibronectin type III-like domain, and a catalytic domain. The N-terminal domain (59 amino acids) was similar to that found in the C-terminal extension of ChiA (50 amino acids) of this strain and furthermore showed significant sequence homology to the regions found in several chitinases and cellulases. Thus, to evaluate the role of the domain, we constructed the hybrid gene that directs the synthesis of the fusion protein with glutathione S-transferase activity. Both the fusion protein and the N-terminal domain itself bound to chitin, indicating that the N-terminal domain of ChiC constitutes an independent chitin-binding domain.

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Chaudhuri, Swarnava, Joseph C. Bruno, Francis Alonzo, Bobbi Xayarath, Nicholas P. Cianciotto, and Nancy E. Freitag. "Contribution of Chitinases to Listeria monocytogenes Pathogenesis." Applied and Environmental Microbiology 76, no. 21 (September 3, 2010): 7302–5. http://dx.doi.org/10.1128/aem.01338-10.

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ABSTRACT Listeria monocytogenes secretes two chitinases and one chitin binding protein. Mutants lacking chiA, chiB, or lmo2467 exhibited normal growth in cultured cells but were defective for growth in the livers and spleens of mice. Mammals lack chitin; thus, L. monocytogenes may have adapted chitinases to recognize alternative substrates to enhance pathogenesis.

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Li, Linyu, Hong Xin, Xialian Xu, Mei Huang, Xinjun Zhang, Yue Chen, Shuping Zhang, Xin-Yuan Fu, and Zhijie Chang. "CHIP Mediates Degradation of Smad Proteins and Potentially Regulates Smad-Induced Transcription." Molecular and Cellular Biology 24, no. 2 (January 15, 2004): 856–64. http://dx.doi.org/10.1128/mcb.24.2.856-864.2004.

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ABSTRACT Transforming growth factor beta (TGF-β)/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-β/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 35S-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.

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Gutiérrez-Román, Martha Ingrid, Michael F. Dunn, Raunel Tinoco-Valencia, Francisco Holguín-Meléndez, Graciela Huerta-Palacios, and Karina Guillén-Navarro. "Potentiation of the synergistic activities of chitinases ChiA, ChiB and ChiC from Serratia marcescens CFFSUR-B2 by chitobiase (Chb) and chitin binding protein (CBP)." World Journal of Microbiology and Biotechnology 30, no. 1 (July 4, 2013): 33–42. http://dx.doi.org/10.1007/s11274-013-1421-2.

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23

Sin, Eun-Jung, Yeon-Kyung Lee, and Young-Soo Sohn. "Detection of IgG Using Thiolated Protein G Modified SPR Sensor Chip." Journal of Sensor Science and Technology 20, no. 6 (November 30, 2011): 434–38. http://dx.doi.org/10.5369/jsst.2011.20.6.434.

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24

Matthews, Brian W. "Protein Science Best Paper awards to Chih-Chia (Jack) Su and Minttu Virkki." Protein Science 24, no. 3 (February 23, 2015): 265–66. http://dx.doi.org/10.1002/pro.2658.

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25

Yamada, Naomi, William K. M. Lai, Nina Farrell, B. Franklin Pugh, and Shaun Mahony. "Characterizing protein–DNA binding event subtypes in ChIP-exo data." Bioinformatics 35, no. 6 (August 28, 2018): 903–13. http://dx.doi.org/10.1093/bioinformatics/bty703.

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Abstract Motivation Regulatory proteins associate with the genome either by directly binding cognate DNA motifs or via protein–protein interactions with other regulators. Each recruitment mechanism may be associated with distinct motifs and may also result in distinct characteristic patterns in high-resolution protein–DNA binding assays. For example, the ChIP-exo protocol precisely characterizes protein–DNA crosslinking patterns by combining chromatin immunoprecipitation (ChIP) with 5′ → 3′ exonuclease digestion. Since different regulatory complexes will result in different protein–DNA crosslinking signatures, analysis of ChIP-exo tag enrichment patterns should enable detection of multiple protein–DNA binding modes for a given regulatory protein. However, current ChIP-exo analysis methods either treat all binding events as being of a uniform type or rely on motifs to cluster binding events into subtypes. Results To systematically detect multiple protein–DNA interaction modes in a single ChIP-exo experiment, we introduce the ChIP-exo mixture model (ChExMix). ChExMix probabilistically models the genomic locations and subtype memberships of binding events using both ChIP-exo tag distribution patterns and DNA motifs. We demonstrate that ChExMix achieves accurate detection and classification of binding event subtypes using in silico mixed ChIP-exo data. We further demonstrate the unique analysis abilities of ChExMix using a collection of ChIP-exo experiments that profile the binding of key transcription factors in MCF-7 cells. In these data, ChExMix identifies possible recruitment mechanisms of FoxA1 and ERα, thus demonstrating that ChExMix can effectively stratify ChIP-exo binding events into biologically meaningful subtypes. Availability and implementation ChExMix is available from https://github.com/seqcode/chexmix. Supplementary information Supplementary data are available at Bioinformatics online.

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26

Lu, Zhen-Xiang, André Laroche, and Hung Chang Huang. "Isolation and characterization of chitinases from Verticillium lecanii." Canadian Journal of Microbiology 51, no. 12 (December 1, 2005): 1045–55. http://dx.doi.org/10.1139/w05-088.

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Degenerate PCR primers corresponding to conserved domains of fungal chitinases were designed, and PCR was performed on genomic DNA of the entomogenous fungus Verticillium lecanii (Zimmermann) Viegas. Two distinct PCR fragments, chf1 and chf2, were isolated and used to identify two DNA contigs. Analyses of these two contigs revealed that we had obtained the full-length DNA sequence including the promoter, 5′ untranslated region, open reading frame (ORF), and 3′ untranslated regions for two distinct chitinase-like genes. These two genomic DNA sequences exhibited 51% identity at the amino acid (aa) level and were designed as acidic (chi1) and basic (chi2) chitinase-like genes. The isolated cDNA for chi1 gene is 1110 bp with a predicted protein of 370 aa and molecular mass of 40.93 kDa, and its ORF was uninterrupted in its corresponding genomic DNA sequence. The cDNA for the chi2 gene is 1269 bp, a predicted ORF of 423 aa and molecular mass of 45.95 kDa. In contrast, the ORF was interrupted by three introns in its corresponding genomic DNA. The basic chitinase gene (chi2) was successfully expressed in the Pichia pastoris system; optimum enzymatic activity was observed at 22 °C and at pH 7.5. CHI1 and CHI2 were clustered into two different phylogenetic groups according to their sequence alignments with 28 other fungal chitinases. A chitin-binding domain, comprising two sub-domains that exhibit similarities at the aa level to chitin binding domains in bacteria, was identified in 30 fungal chitinase sequences examined.Key words: fungus, chitin, cloning, sequencing, transformation, Pichia sp. expression.

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Rodriguez, Benjamin A. T., and Tim H. M. Huang. "Tilling the chromatin landscape: emerging methods for the discovery and profiling of protein–DNA interactions." Biochemistry and Cell Biology 83, no. 4 (August 1, 2005): 525–34. http://dx.doi.org/10.1139/o05-055.

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Interactions between protein and DNA are essential for cellular function. The incremental process of developing global approaches to study chromatin began with the in vitro characterization of chromatin structural components and modifications of the versatile chromatin immunoprecipitation (ChIP) assay, capable of analyzing protein–DNA interactions in vivo. Among the emerging global approaches are ChIP cloning, ChIP display, differential chromatin scanning, ChIP–chip, DamID chromatin profiling, and chromatin array. These methods have been used to assess transcription-factor binding and (or) histone modification. This review describes these global methods and illustrates their potential in answering biological questions.Key words: ChIP, transcription factor binding, histone modification, ChIP display, differential chromatin scanning, ChIP-chip, DamID chromatin profiling, chromatin array.

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Xia, Tian, Christiana Dimitropoulou, Jingmin Zeng, Galina N. Antonova, Connie Snead, Richard C. Venema, David Fulton, et al. "Chaperone-dependent E3 ligase CHIP ubiquitinates and mediates proteasomal degradation of soluble guanylyl cyclase." American Journal of Physiology-Heart and Circulatory Physiology 293, no. 5 (November 2007): H3080—H3087. http://dx.doi.org/10.1152/ajpheart.00579.2007.

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The nitric oxide receptor soluble guanylyl cyclase (sGC) exists in multimeric protein complexes, including heat shock protein (HSP) 90 and endothelial nitric oxide synthase. Inhibition of HSP90 by geldanamycin causes proteasomal degradation of sGC protein. In this study, we have investigated whether COOH terminus of heat shock protein 70-interacting protein (CHIP), a co-chaperone molecule that is involved in protein folding but is also a chaperone-dependent ubiquitin E3 ligase, could play a role in the process of degradation of sGC. Transient overexpression of CHIP in COS-7 cells degraded heterologous sGC in a concentration-related manner; this downregulation of sGC was abrogated by the proteasome inhibitor MG-132. Transfection of tetratricopeptide repeats and U-box domain CHIP mutants attenuated sGC degradation, suggesting that both domains are indispensable for CHIP function. Results from immunoprecipitation and indirect immunofluorescent microscopy experiments demonstrated that CHIP is associated with sGC, HSP90, and HSP70 in COS-7 cells. Furthermore, CHIP increased the association of HSP70 with sGC. In in vitro ubiquitination assays using purified proteins and ubiquitin enzymes, E3 ligase CHIP directly ubiquitinated sGC; this ubiquitination was potentiated by geldanamycin in COS-7 cells, followed by proteasomal degradation. In rat aortic smooth muscle cells, endogenous sGC was also degraded by adenovirus-infected wild-type CHIP but not by the chaperone interaction-deficient K30A CHIP, whereas CHIP, but not K30A, attenuated sGC expression in, and nitric oxide donor-induced relaxation of, rat aortic rings, suggesting that CHIP plays a regulatory role under physiological conditions. This study reveals a new mechanism for the regulation of sGC, an important mediator of cellular and vascular function.

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Min, Jin-Na, Ryan A. Whaley, Norman E. Sharpless, Pamela Lockyer, Andrea L. Portbury, and Cam Patterson. "CHIP Deficiency Decreases Longevity, with Accelerated Aging Phenotypes Accompanied by Altered Protein Quality Control." Molecular and Cellular Biology 28, no. 12 (April 14, 2008): 4018–25. http://dx.doi.org/10.1128/mcb.00296-08.

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ABSTRACT During the course of biological aging, there is a gradual accumulation of damaged proteins and a concomitant functional decline in the protein degradation system. Protein quality control is normally ensured by the coordinated actions of molecular chaperones and the protein degradation system that collectively help to maintain protein homeostasis. The carboxyl terminus of Hsp70-interacting protein (CHIP), a ubiquitin ligase/cochaperone, participates in protein quality control by targeting a broad range of chaperone substrates for proteasome degradation via the ubiquitin-proteasome system, demonstrating a broad involvement of CHIP in maintaining cytoplasmic protein quality control. In the present study, we have investigated the influence that protein quality control exerts on the aging process by using CHIP−/− mice. CHIP deficiency in mice leads to a markedly reduced life span, along with accelerated age-related pathophysiological phenotypes. These features were accompanied by indications of accelerated cellular senescence and increased indices of oxidative stress. In addition, CHIP−/− mice exhibit a deregulation of protein quality control, as indicated by elevated levels of toxic oligomer proteins and a decline in proteasome activity. Taken together, these data reveal that impaired protein quality control contributes to cellular senescence and implicates CHIP-dependent quality control mechanisms in the regulation of mammalian longevity in vivo.

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Huang, Zhong, Lei Nie, Min Xu, and Xiao-Hong Sun. "Notch-Induced E2A Degradation Requires CHIP and Hsc70 as Novel Facilitators of Ubiquitination." Molecular and Cellular Biology 24, no. 20 (October 15, 2004): 8951–62. http://dx.doi.org/10.1128/mcb.24.20.8951-8962.2004.

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ABSTRACT 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.

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Chen, Xing, Da Fu Cui, Lu Lu Zhang, Hui Li, Jian Hai Sun, and Hao Yuan Cai. "A Porous Microfluidic Chip for Protein Extraction Based on Solid Phase Extraction Method." Key Engineering Materials 483 (June 2011): 297–300. http://dx.doi.org/10.4028/www.scientific.net/kem.483.297.

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Based on the principle of solid phase extraction (SPE) and the special immunoreaction, a microfluidic chip integrated with porous matrix was developed for protein extraction. Porous matrix was achieved by electrochemical etching silicon in a HF/ethanol mixture, which was coated on the wall of the rectangular channel of the microchip to provide a surface-enlarging matrix. The surface morphology of the bare porous silicon and the porous silicon modified with the protein has been characterized by SEM. Non-porous chip and porous chip were used to extract protein. Compared with non-porous matrix, the porous matrix achieved higher extracted efficiency of protein. Then two methods of surface modification were employed on porous matrix for protein extraction. The surface modification with Protein A could extract more protein with less non-special absorption. Evaluation of the structure of the solid phase matrix and the surface modification process in the microfluidic chip, the porous microfluidic chip is able to be suitable for incorporation into micro total analysis system (μTAS).

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Li, Xueni, Mei Huang, Huiling Zheng, Yinyin Wang, Fangli Ren, Yu Shang, Yonggong Zhai, et al. "CHIP promotes Runx2 degradation and negatively regulates osteoblast differentiation." Journal of Cell Biology 181, no. 6 (June 9, 2008): 959–72. http://dx.doi.org/10.1083/jcb.200711044.

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Runx2, an essential transactivator for osteoblast differentiation, is tightly regulated at both the transcriptional and posttranslational levels. In this paper, we report that CHIP (C terminus of Hsc70-interacting protein)/STUB1 regulates Runx2 protein stability via a ubiquitination-degradation mechanism. CHIP interacts with Runx2 in vitro and in vivo. In the presence of increased Runx2 protein levels, CHIP expression decreases, whereas the expression of other E3 ligases involved in Runx2 degradation, such as Smurf1 or WWP1, remains constant or increases during osteoblast differentiation. Depletion of CHIP results in the stabilization of Runx2, enhances Runx2-mediated transcriptional activation, and promotes osteoblast differentiation in primary calvarial cells. In contrast, CHIP overexpression in preosteoblasts causes Runx2 degradation, inhibits osteoblast differentiation, and instead enhances adipogenesis. Our data suggest that negative regulation of the Runx2 protein by CHIP is critical in the commitment of precursor cells to differentiate into the osteoblast lineage.

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Ballinger, Carol A., Patrice Connell, Yaxu Wu, Zhaoyong Hu, Larry J. Thompson, Li-Yan Yin, and Cam Patterson. "Identification of CHIP, a Novel Tetratricopeptide Repeat-Containing Protein That Interacts with Heat Shock Proteins and Negatively Regulates Chaperone Functions." Molecular and Cellular Biology 19, no. 6 (June 1, 1999): 4535–45. http://dx.doi.org/10.1128/mcb.19.6.4535.

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ABSTRACT 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.

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Liao, Wei. "Novel probes for protein chip applications." Frontiers in Bioscience 11, no. 1 (2006): 186. http://dx.doi.org/10.2741/1790.

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35

Sheridan, Cormac. "Protein chip companies turn to biomarkers." Nature Biotechnology 23, no. 1 (January 2005): 3–4. http://dx.doi.org/10.1038/nbt0105-3.

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Sandison, Mairi E., Sarah A. Cumming, Walter Kolch, and Andrew R. Pitt. "On-chip immunoprecipitation for protein purification." Lab on a Chip 10, no. 20 (2010): 2805. http://dx.doi.org/10.1039/c005295g.

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Caputo, Emilia, Ramy Moharram, and Brian M. Martin. "Methods for on-chip protein analysis." Analytical Biochemistry 321, no. 1 (October 2003): 116–24. http://dx.doi.org/10.1016/s0003-2697(03)00361-0.

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Senior, Kathryn. "Fingerprinting disease with protein chip arrays." Molecular Medicine Today 5, no. 8 (August 1999): 326–27. http://dx.doi.org/10.1016/s1357-4310(99)01536-1.

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Jain, Shubha, Sarpras Swain, Lopamudra Das, Sarita Swain, Lopamudra Giri, Anand Kumar Kondapi, and Harikrishnan Narayanan Unni. "Microfluidic Protein Imaging Platform: Study of Tau Protein Aggregation and Alzheimer’s Drug Response." Bioengineering 7, no. 4 (December 13, 2020): 162. http://dx.doi.org/10.3390/bioengineering7040162.

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Tau protein aggregation is identified as one of the key phenomena associated with the onset and progression of Alzheimer’s disease. In the present study, we performed on-chip confocal imaging of tau protein aggregation and tau–drug interactions using a spiral-shaped passive micromixing platform. Numerical simulations and experiments were performed in order to validate the performance of the micromixer design. We performed molecular modeling of adenosine triphosphate (ATP)-induced tau aggregation in order to successfully validate the concept of helical tau filament formation. Tau aggregation and native tau restoration were realized using an immunofluorescence antibody assay. The dose–response behavior of an Alzheimer’s drug, methylthioninium chloride (MTC), was monitored on-chip for defining the optimum concentration of the drug. The proposed device was tested for reliability and repeatability of on-chip tau imaging. The amount of the tau protein sample used in our experiments was significantly less than the usage for conventional techniques, and the whole protein–drug assay was realized in less than two hours. We identified that intensity-based tau imaging could be used to study Alzheimer’s drug response. In addition, it was demonstrated that cell-free, microfluidic tau protein assays could be used as potential on-chip drug evaluation tools for Alzheimer’s disease.

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Baratto, César Milton, Marcia Vanusa da Silva, Lucélia Santi, Luciane Passaglia, Irene Silveira Schrank, Marilene Henning Vainstein, and Augusto Schrank. "Expression and characterization of the 42 kDa chitinase of the biocontrol fungusMetarhizium anisopliaeinEscherichia coli." Canadian Journal of Microbiology 49, no. 11 (November 1, 2003): 723–26. http://dx.doi.org/10.1139/w03-085.

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Albeit Metarhizium anisopliae is the best-characterized entomopathogenic fungus, the role of some hydrolytic enzymes during host cuticle penetration has not yet been established. Three chitinase genes (chit1, chi2, chi3) from Metarhizium have already been isolated. To characterize the chitinase coded by the chit1 gene, we expressed the active protein (CHIT42) in Escherichia coli using a T7-based promoter expression vector. The recombinant protein, CHIT42, is active against glycol chitin and synthetic N-acetylglucosamine (GlcNAc) dimer and tetramer substrates. These activities suggest that the recombinant CHIT42 acts as an endochitinase.Key words: Metarhizium anisopliae, chitinases, chit genes, recombinant protein, enthomopathogenic fungi.

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Arndt, Verena, Christina Daniel, Wolfgang Nastainczyk, Simon Alberti, and Jörg Höhfeld. "BAG-2 Acts as an Inhibitor of the Chaperone-associated Ubiquitin Ligase CHIP." Molecular Biology of the Cell 16, no. 12 (December 2005): 5891–900. http://dx.doi.org/10.1091/mbc.e05-07-0660.

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Cellular protein quality control involves a close interplay between molecular chaperones and the ubiquitin/proteasome system. We recently identified a degradation pathway, on which the chaperone Hsc70 delivers chaperone clients, such as misfolded forms of the cystic fibrosis transmembrane conductance regulator (CFTR), to the proteasome. The cochaperone CHIP is of central importance on this pathway, because it acts as a chaperone-associated ubiquitin ligase. CHIP mediates the attachment of a ubiquitin chain to a chaperone-presented client protein and thereby stimulates its proteasomal degradation. To gain further insight into the function of CHIP we isolated CHIP-containing protein complexes from human HeLa cells and analyzed their composition by peptide mass fingerprinting. We identified the Hsc70 cochaperone BAG-2 as a main component of CHIP complexes. BAG-2 inhibits the ubiquitin ligase activity of CHIP by abrogating the CHIP/E2 cooperation and stimulates the chaperone-assisted maturation of CFTR. The activity of BAG-2 resembles that of the previously characterized Hsc70 cochaperone and CHIP inhibitor HspBP1. The presented data therefore establish multiple mechanisms to control the destructive activity of the CHIP ubiquitin ligase in human cells.

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Sha, Youbao, Lavannya Pandit, Shenyan Zeng, and N. Tony Eissa. "A Critical Role for CHIP in the Aggresome Pathway." Molecular and Cellular Biology 29, no. 1 (October 27, 2008): 116–28. http://dx.doi.org/10.1128/mcb.00829-08.

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ABSTRACT Recent evidence suggests that aggresome formation is a physiologic stress response not limited to misfolded proteins. That stress response, termed “physiologic aggresome,” is exemplified by aggresome formation of inducible nitric oxide synthase (iNOS), an important host defense protein. CHIP (carboxy terminus of Hsp70-interacting protein) is a highly conserved protein that has been shown to mediate substrate ubiquitination and degradation by the proteasome. In this study, we show that CHIP has a previously unexpected critical role in the aggresome pathway. CHIP interacts with iNOS and promotes its ubiquitination and degradation by the proteasome as well as its sequestration to the aggresome. CHIP-mediated iNOS targeting to the proteasome sequentially precedes CHIP-mediated iNOS sequestration to the aggresome. CHIP is required for iNOS preaggresome structures to form a mature aggresome. Furthermore, CHIP is required for targeting the mutant form of cystic fibrosis transconductance regulator (CFTRΔF508) to the aggresome. Importantly, the ubiquitin ligase function of CHIP is required in targeting preaggresomal structures to the aggresome by promoting an iNOS interaction with histone deacetylase 6, which serves as an adaptor between ubiquitinated proteins and the dynein motor. This study reveals a critical role for CHIP in the aggresome pathway.

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Xu, Jing, Jia-yu Wan, Song-tao Yang, Shou-feng Zhang, Na Xu, Nan Li, Ji-ping Li, Hai-ying Wang, Xue Bai, and Wen-sen Liu. "A surface plasmon resonance biosensor for direct detection of the rabies virus." Acta Veterinaria Brno 81, no. 2 (2012): 107–11. http://dx.doi.org/10.2754/avb201281020107.

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A surface plasmon resonance biosensor chip was constructed for detection of rabies virus. For the construction of the biosensor chip, N protein specific antibody and N protein specific antibody combined with G protein specific antibody of rabies virus were linked on two different flow cells on one CM5 chip, respectively. The chip was tested for the detection of rabies virus antigens using the crude extract of rabies virus from infected BHK cell strain culture. Tenfold serial dilutions of SRV9 strain virus-infected cell cultures were tested by the biosensor chip to establish the detection limit. The limit detection was approximately 70 pg/ml of nucleoprotein and glycoprotein. The biosensor chip developed in this study was employed for the detection of rabies virus in five suspect infectious specimens of brain tissue from guinea pigs; the results were compared by fluorescent antibody test. Surface plasmon resonance biosensor chip could be a useful automatic tool for prompt detection of rabies virus infection.

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Yun, Choong-Soo, Chiho Suzuki, Kunihiko Naito, Toshiharu Takeda, Yurika Takahashi, Fumiya Sai, Tsuguno Terabayashi, et al. "Pmr, a Histone-Like Protein H1 (H-NS) Family Protein Encoded by the IncP-7 Plasmid pCAR1, Is a Key Global Regulator That Alters Host Function." Journal of Bacteriology 192, no. 18 (July 16, 2010): 4720–31. http://dx.doi.org/10.1128/jb.00591-10.

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ABSTRACT Histone-like protein H1 (H-NS) family proteins are nucleoid-associated proteins (NAPs) conserved among many bacterial species. The IncP-7 plasmid pCAR1 is transmissible among various Pseudomonas strains and carries a gene encoding the H-NS family protein, Pmr. Pseudomonas putida KT2440 is a host of pCAR1, which harbors five genes encoding the H-NS family proteins PP_1366 (TurA), PP_3765 (TurB), PP_0017 (TurC), PP_3693 (TurD), and PP_2947 (TurE). Quantitative reverse transcription-PCR (qRT-PCR) demonstrated that the presence of pCAR1 does not affect the transcription of these five genes and that only pmr, turA, and turB were primarily transcribed in KT2440(pCAR1). In vitro pull-down assays revealed that Pmr strongly interacted with itself and with TurA, TurB, and TurE. Transcriptome comparisons of the pmr disruptant, KT2440, and KT2440(pCAR1) strains indicated that pmr disruption had greater effects on the host transcriptome than did pCAR1 carriage. The transcriptional levels of some genes that increased with pCAR1 carriage, such as the mexEF-oprN efflux pump genes and parI, reverted with pmr disruption to levels in pCAR1-free KT2440. Transcriptional levels of putative horizontally acquired host genes were not altered by pCAR1 carriage but were altered by pmr disruption. Identification of genome-wide Pmr binding sites by ChAP-chip (chromatin affinity purification coupled with high-density tiling chip) analysis demonstrated that Pmr preferentially binds to horizontally acquired DNA regions. The Pmr binding sites overlapped well with the location of the genes differentially transcribed following pmr disruption on both the plasmid and the chromosome. Our findings indicate that Pmr is a key factor in optimizing gene transcription on pCAR1 and the host chromosome.

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Bukharina, N. S., Yu D. Ivanov, T. O. Pleshakova, P. A. Frantsuzov, E. Yu Andreeva, A. L. Kaysheva, A. A. Izotov, et al. "Atomic force microscopy fishing of gp120 on immobilized aptamer and its mass spectrometry identification." Biomeditsinskaya Khimiya 61, no. 3 (2015): 363–72. http://dx.doi.org/10.18097/pbmc20156103363.

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A method of atomic force microscopy-based fishing (AFM fishing) has been developed for protein detection in the analyte solution using a chip with an immobilized aptamer. This method is based on the biospecific fishing of a target protein from a bulk solution onto the small AFM chip area with the immobilized aptamer to this protein used as the molecular probe. Such aptamer-based approach allows to increase an AFM image contrast compared to the antibody-based approach. Mass spectrometry analysis used after the biospecific fishing to identify the target protein on the AFM chip has proved complex formation. Use of the AFM chip with the immobilized aptamer avoids interference of the antibody and target protein peaks in a mass spectrum.

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46

Ravi, Saranya, Traci Parry, Monte Willis, Pamela Lockyer, Cam Patterson, James Bain, Robert Stevens, Olga Ilkayeva, Christopher Newgard, and Jonathan Schisler. "Adverse Effects of Fenofibrate in Mice Deficient in the Protein Quality Control Regulator, CHIP." Journal of Cardiovascular Development and Disease 5, no. 3 (August 15, 2018): 43. http://dx.doi.org/10.3390/jcdd5030043.

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We previously reported how the loss of CHIP expression (Carboxyl terminus of Hsc70-Interacting Protein) during pressure overload resulted in robust cardiac dysfunction, which was accompanied by a failure to maintain ATP levels in the face of increased energy demand. In this study, we analyzed the cardiac metabolome after seven days of pressure overload and found an increase in long-chain and medium-chain fatty acid metabolites in wild-type hearts. This response was attenuated in mice that lack expression of CHIP (CHIP−/−). These findings suggest that CHIP may play an essential role in regulating oxidative metabolism pathways that are regulated, in part, by the nuclear receptor PPARα (Peroxisome Proliferator-Activated Receptor alpha). Next, we challenged CHIP−/− mice with the PPARα agonist called fenofibrate. We found that treating CHIP−/− mice with fenofibrate for five weeks under non-pressure overload conditions resulted in decreased skeletal muscle mass, compared to wild-type mice, and a marked increase in cardiac fibrosis accompanied by a decrease in cardiac function. Fenofibrate resulted in decreased mitochondrial cristae density in CHIP−/− hearts as well as decreased expression of genes involved in the initiation of autophagy and mitophagy, which suggests that a metabolic challenge, in the absence of CHIP expression, impacts pathways that contribute to mitochondrial quality control. In conclusion, in the absence of functional CHIP expression, fenofibrate results in unexpected skeletal muscle and cardiac pathologies. These findings are particularly relevant to patients harboring loss-of-function mutations in CHIP and are consistent with a prominent role for CHIP in regulating cardiac metabolism.

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47

ZHANG, YONG. "INTEGRATION OF NANOPARTICLES WITH PROTEIN MICROARRAYS." International Journal of Nanoscience 05, no. 02n03 (April 2006): 189–94. http://dx.doi.org/10.1142/s0219581x0600422x.

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A variety of DNA, protein or cell microarray devices and systems have been developed and commercialized. In addition to the biomolecule related analysis, they are also being used for pharmacogenomic research, infectious and genetic disease and cancer diagnostics, and proteomic and cellular analysis.1 Currently, microarray is fabricated on a planar surface; this limits the amount of biomolecules that can be bounded on the surface. In this work, a planar protein microarray chip with nonplanar spot surface was fabricated to enhance the chip performance. A nonplanar spot surface was created by first coating the silica nanoparticles with albumin and depositing them into the patterned microwells. The curve surfaces of the nanoparticles increase the surface area for immobilization of proteins, which helps to enhance the detection sensitivity of the chip. Using this technique, proteins are immobilized onto the nanoparticles before they are deposited onto the chip, and therefore the method of protein immobilization can be customized at each spot. Furthermore, a nonplanar surface promotes the retention of native protein structure better than planar surface.2 The technique developed can be used to produce different types of microarrays, such as DNA, protein and antibody microarrays.

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48

Busque, Lambert, Maxine Sun, Manuel Buscarlet, Sami Ayachi, Yassamin Feroz Zada, Sylvie Provost, Vincent Bourgoin, et al. "High-sensitivity C-reactive protein is associated with clonal hematopoiesis of indeterminate potential." Blood Advances 4, no. 11 (June 3, 2020): 2430–38. http://dx.doi.org/10.1182/bloodadvances.2019000770.

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Abstract Clonal hematopoiesis of indeterminate potential (CHIP) is predictive of hematological cancers and cardiovascular diseases, but the etiology of CHIP initiation and clonal expansion is unknown. Several lines of evidence suggest that proinflammatory cytokines may favor mutated hematopoietic stem cell expansion. To investigate the potential link between inflammation and CHIP, we performed targeted deep sequencing of 11 genes previously implicated in CHIP in 1887 subjects aged >70 years from the Montreal Heart Institute Biobank, of which 1359 had prior coronary artery disease (CAD), and 528 controls did not. We assessed association of CHIP with log transformed high-sensitivity C-reactive protein (hs-CRP), a validated biomarker of inflammation. CHIP was identified in 427 of the 1887 subjects (22.6%). CHIP mutations were more frequently identified in DNMT3A (11.6%) and TET2 (6.1%), with a higher proportion of TET2 mutations occurring in controls than in patients with CAD (9.0% vs 4.9%, P < .001). CHIP carriers had 21% higher hs-CRP levels compared with their noncarrier counterparts (eβ = 1.21, 95% confidence interval [CI]: 1.08 to 1.36; P = .001). A similar effect was observed in the subgroup of patients with known CAD (eβ = 1.22, 95% CI: 1.06 to 1.41; P = .005). These findings confirm the association between inflammation and CHIP. This association may open investigational avenues aimed at documenting mechanisms linking inflammation to clonal progression and ultimately supports prevention interventions to attenuate CHIP’s impact on cardiovascular disease and cancer.

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Yang, Mingjin, Chen Wang, Xuhui Zhu, Songqing Tang, Liyun Shi, Xuetao Cao та Taoyong Chen. "E3 ubiquitin ligase CHIP facilitates Toll-like receptor signaling by recruiting and polyubiquitinating Src and atypical PKCζ". Journal of Experimental Medicine 208, № 10 (12 вересня 2011): 2099–112. http://dx.doi.org/10.1084/jem.20102667.

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The carboxyl terminus of constitutive heat shock cognate 70 (HSC70)–interacting protein (CHIP, also known as Stub1) is a U box–containing E3 ubiquitin ligase that is important for protein quality control. The role of CHIP in innate immunity is not known. Here, we report that CHIP knockdown inhibits Toll-like receptor (TLR) 4– and TLR9-driven signaling, but not TLR3-driven signaling; proinflammatory cytokine and type 1 interferon (IFN) production; and maturation of antigen-presenting cells, including macrophages and dendritic cells. We demonstrate that CHIP can recruit the tyrosine kinase Src and atypical protein kinase C ζ (PKCζ) to the TLR complex, thereby leading to activation of IL-1 receptor–associated kinase 1, TANK-binding kinase 1, and IFN regulatory factors 3 and 7. CHIP acts as an E3 ligase for Src and PKCζ during TLR signaling. CHIP-mediated enhancement of TLR signaling is inhibited by IFNAR deficiency or expression of ubiquitination resistant mutant forms of Src or PKCζ. These findings suggest that CHIP facilitates the formation of a TLR signaling complex by recruiting, ubiquitinating, and activating Src and PKCζ.

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Vidal, Roberto M., David A. Montero, Felipe Del Canto, Juan C. Salazar, Carolina Arellano, Alhejandra Alvarez, Nora L. Padola, et al. "Safety and Immunogenicity of a Chimeric Subunit Vaccine against Shiga Toxin-Producing Escherichia coli in Pregnant Cows." International Journal of Molecular Sciences 24, no. 3 (February 1, 2023): 2771. http://dx.doi.org/10.3390/ijms24032771.

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Shiga toxin-producing Escherichia coli (STEC) is a zoonotic pathogen that causes gastroenteritis and Hemolytic Uremic Syndrome. Cattle are the main animal reservoir, excreting the bacteria in their feces and contaminating the environment. In addition, meat can be contaminated by releasing the intestinal content during slaughtering. Here, we evaluated the safety and immunogenicity of a vaccine candidate against STEC that was formulated with two chimeric proteins (Chi1 and Chi2), which contain epitopes of the OmpT, Cah and Hes proteins. Thirty pregnant cows in their third trimester of gestation were included and distributed into six groups (n = 5 per group): four groups were administered intramuscularly with three doses of the formulation containing 40 µg or 100 µg of each protein plus the Quil-A or Montanide™ Gel adjuvants, while two control groups were administered with placebos. No local or systemic adverse effects were observed during the study, and hematological parameters and values of blood biochemical indicators were similar among all groups. Furthermore, all vaccine formulations triggered systemic anti-Chi1/Chi2 IgG antibody levels that were significantly higher than the control groups. However, specific IgA levels were generally low and without significant differences among groups. Notably, anti-Chi1/Chi2 IgG antibody levels in the serum of newborn calves fed with colostrum from their immunized dams were significantly higher compared to newborn calves fed with colostrum from control cows, suggesting a passive immunization through colostrum. These results demonstrate that this vaccine is safe and immunogenic when applied to pregnant cows during the third trimester of gestation.

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