scholarly journals The coiled coil of in vitro assembled keratin filaments is a heterodimer of type I and II keratins: use of site-specific mutagenesis and recombinant protein expression.

1990 ◽  
Vol 110 (4) ◽  
pp. 1199-1210 ◽  
Author(s):  
M Hatzfeld ◽  
K Weber
Keyword(s):  
Recombinant Dna ◽  
Recombinant Protein Expression ◽  
Coiled Coil ◽  
Heptad Repeat ◽  
In Vitro Mutagenesis ◽  
Type I ◽  
Air Oxidation ◽  
Cross Link ◽  
Site Specific

Recombinant DNA technology has been used to analyze the first step in keratin intermediate filament (IF) assembly; i.e., the formation of the double stranded coiled coil. Keratins 8 and 18, lacking cysteine, were subjected to site specific in vitro mutagenesis to change one amino acid in the same relative position of the alpha-helical rod domain of both keratins to a cysteine. The mutations lie at position -36 of the rod in a "d" position of the heptad repeat pattern, and thus air oxidation can introduce a zero-length cystine cross-link. Mutant keratins 8 and 18 purified separately from Escherichia coli readily formed cystine homodimers in 2 M guanidine-HCl, and could be separated from the monomers by gel filtration. Heterodimers with a cystine cross-link were obtained when filaments formed by the two reduced monomers were allowed to oxidize. Subsequent ion exchange chromatography in 8.5 M urea showed that only a single dimer species had formed. Diagonal electrophoresis and reverse phase HPLC identified the dimer as the cystine containing heterodimer. This heterodimer readily assembled again into IF indistinguishable from those obtained from the nonmutant counterparts or from authentic keratins. In contrast, the mixture of cystine-stabilized homodimers formed only large aberrant aggregates. However, when a reducing agent was added, filaments formed again and yielded the heterodimer after oxidation. Thus, the obligatory heteropolymer step in keratin IF assembly seems to occur preferentially at the dimer level and not during tetramer formation. Our results also suggest that keratin I and II homodimers, once formed, are at least in 2 M guanidine-HCl a metastable species as their mixtures convert spontaneously into heterodimers unless the homodimers are stabilized by the cystine cross-link. This previously unexpected property of homodimers explains major discrepancies in the literature on the keratin dimer.

scholarly journals Site-specific ubiquitination exposes a linear motif to promote interferon-α receptor endocytosis

2007 ◽  
Vol 179 (5) ◽  
pp. 935-950 ◽  
Author(s):  
K.G. Suresh Kumar ◽  
Hervé Barriere ◽  
Christopher J. Carbone ◽  
Jianghuai Liu ◽  
Gayathri Swaminathan ◽  
...  
Keyword(s):  
Ubiquitin Ligase ◽  
E3 Ubiquitin Ligase ◽  
Interferon Α ◽  
Type I ◽  
Lysosomal Degradation ◽  
Linear Motif ◽  
Site Specific ◽  
Receptor Endocytosis ◽  
Β Receptor

Ligand-induced endocytosis and lysosomal degradation of cognate receptors regulate the extent of cell signaling. Along with linear endocytic motifs that recruit the adaptin protein complex 2 (AP2)–clathrin molecules, monoubiquitination of receptors has emerged as a major endocytic signal. By investigating ubiquitin-dependent lysosomal degradation of the interferon (IFN)-α/β receptor 1 (IFNAR1) subunit of the type I IFN receptor, we reveal that IFNAR1 is polyubiquitinated via both Lys48- and Lys63-linked chains. The SCFβTrcp (Skp1–Cullin1–F-box complex) E3 ubiquitin ligase that mediates IFNAR1 ubiquitination and degradation in cells can conjugate both types of chains in vitro. Although either polyubiquitin linkage suffices for postinternalization sorting, both types of chains are necessary but not sufficient for robust IFNAR1 turnover and internalization. These processes also depend on the proximity of ubiquitin-acceptor lysines to a linear endocytic motif and on its integrity. Furthermore, ubiquitination of IFNAR1 promotes its interaction with the AP2 adaptin complex that is required for the robust internalization of IFNAR1, implicating cooperation between site-specific ubiquitination and the linear endocytic motif in regulating this process.

scholarly journals P029 TRIM 21 protects against ulcerative colitis and colitic cancer via smad7 inhibition

2020 ◽  
Vol 14 (Supplement_1) ◽  
pp. S146-S146
Author(s):  
K B Hahm
Keyword(s):  
Ulcerative Colitis ◽  
Coiled Coil ◽  
Type I ◽  
Typical Structure ◽  
Th17 Cell Differentiation ◽  
Bowel Diseases ◽  
Colitic Cancer ◽  
Β Receptor ◽  
Trim Proteins

Abstract Background Proteins of the tripartite motif-containing (TRIM) superfamily are critical in a variety of biological processes in either innate immunity or eliminating invading pathogens, by which had been implicated in pathogenesis of autoimmune diseases including inflammatory bowel diseases. The typical structure of TRIM proteins contains a RING motif in the N-terminal end, followed by a B-box motif, a coiled-coil domain and a B30.2/PRYSPRY region in the C-terminal end led to the regulation of TGF-β anti-inflammatory cytokines, by which TRIM21 has been reported to regulate IBD negatively through inhibiting Th1/Th17 cell differentiation. Methods Since antisense oligonucleotide targeting smad7 was withdrawn from clinical trial due to insufficient efficacy, in this study, we generated TRIM21 overexpressed cell lines to study the binding of TRIM21 to smad7 as well as the regulation of consequent TGF-β receptor. Results TRIM21 significantly binds to smad7 as well as repressed levels of TGF-b type I/II receptor. SBE-luc and 3TP-luc assay showed significantly decreased activities under TRIM21 + TGF-β. Since TRIM21 contains ubiquitin ligase, PRYSPRY, TRIM21 with TGF-β significantly decreased TGFRII via UPL. These in vitro evidences that TRIM21 significantly repressed TGF-β after binding smad7 were validated with DSS-induced colitis and colitic cancer model. TRIM21 was significantly decreased in DSS-induced ulcerative colitis, whereas ameliorated colitis showed significant restoration of TRIM21 Conclusion Leading to conclusion that loss of TRIM21 led to significant bout of IBD.

scholarly journals Newt satellite 2 transcripts self-cleave by using an extended hammerhead structure.

1991 ◽  
Vol 11 (12) ◽  
pp. 6109-6115 ◽  
Author(s):  
L M Pabón-Peña ◽  
Y Zhang ◽  
L M Epstein
Keyword(s):  
Structural Model ◽  
In Vitro Mutagenesis ◽  
Base Pairs ◽  
Site Specific ◽  
Internal Loops

Synthetic transcripts of satellite 2 DNA from newts undergo self-catalyzed, site-specific cleavage in vitro. Cleavage occurs within a domain that is similar to the hammerhead domain used by a number of self-cleaving, infectious plant RNAs. The newt hammerhead has a potentially unstable structure due to a stem composed of two base pairs and a 2-nucleotide loop, and unlike other hammerheads that have been studied, it cannot cleave as an isolated unit. Here we show that cleavage by a single newt hammerhead requires additional satellite 2 sequences flanking both ends of the hammerhead domain. We also present a structural model of a truncated satellite 2 transcript which is capable of cleavage. The structure includes an internally looped extension to one of the conserved stems of the hammerhead. By in vitro mutagenesis, the identities of each of the five nucleotides composing one of the internal loops were shown to be critical for cleavage. Additional evidence that the extension stimulates self-cleavage in a manner other than by simply stabilizing the hammerhead is presented.

scholarly journals Identification of protein IT of the intestinal cytoskeleton as a novel type I cytokeratin with unusual properties and expression patterns.

1990 ◽  
Vol 111 (2) ◽  
pp. 567-580 ◽  
Author(s):  
R Moll ◽  
D L Schiller ◽  
W W Franke
Keyword(s):  
Mammalian Species ◽  
Expression Patterns ◽  
Prostate Gland ◽  
Coiled Coil ◽  
Immunological Methods ◽  
Type I ◽  
Peptide Fragments ◽  
Binding Assays ◽  
Human Intestinal Epithelium

A major cytoskeletal polypeptide (Mr approximately 46,000; protein IT) of human intestinal epithelium was characterized by biochemical and immunological methods. The polypeptide, which was identified as a specific and genuine mRNA product by translation in vitro, reacted, in immunoblotting after SDS-PAGE, only with one of numerous cytokeratin (CK) antisera tested but with none of many monoclonal CK antibodies. In vitro, it formed heterotypic complexes with the type II CK 8, as shown by blot binding assays and gel electrophoresis in 4 M urea, and these complexes assembled into intermediate filaments (IFs) under appropriate conditions. A chymotrypsin-resistant Mr approximately 38,000 core fragment of protein IT could be obtained from cytoskeletal IFs, indicating its inclusion in a coiled coil. Antibodies raised against protein IT decorated typical CK fibril arrays in normal and transformed intestinal cells. Four proteolytic peptide fragments obtained from purified polypeptide IT exhibited significant amino acid sequence homology with corresponding regions of coils I and II of the rod domain of several other type I CKs. Immunocytochemically, the protein was specifically detected as a prominent component of intestinal and gastric foveolar epithelium, urothelial umbrella cells, and Merkel cells of epidermis. Sparse positive epithelial cells were noted in the thymus, bronchus, gall bladder, and prostate gland. The expression of protein IT was generally maintained in primary and metastatic colorectal carcinomas as well as in cell cultures derived therefrom. A corresponding protein was also found in several other mammalian species. We conclude that polypeptide IT is an integral IF component which is related, though somewhat distantly, to type I CKs, and, therefore, we propose to add it to the human CK catalogue as CK 20.

scholarly journals Tailless keratins assemble into regular intermediate filaments in vitro

1990 ◽  
Vol 97 (2) ◽  
pp. 317-324
Author(s):  
M. Hatzfeld ◽  
K. Weber
Keyword(s):  
Intermediate Filaments ◽  
Consensus Sequence ◽  
In Vitro Mutagenesis ◽  
Type I ◽  
Type Ii ◽  
Tail Domain ◽  
Filament Formation ◽  
Wild Type ◽  
Keratin 8

To study the influence of the non alpha-helical tail domain of keratins in filament formation, we prepared a truncated keratin 8 mutant, K8/tailless. Using site-directed in vitro mutagenesis we introduced a stop codon in the position coding for amino acid number 417 of the K8/wild-type sequence, thereby deleting 86 amino acids of the non alpha-helical tail domain but leaving the consensus sequence at the end of the rod domain intact. Expression of the truncated keratin 8 in Escherichia coli allowed us to purify the protein by a two-step procedure. The filament-forming capacity of the truncated K8 with wild-type K18 and K19 was analyzed using in vitro reconstitution. The in vitro assembly studies with K8/tailless and K18 wild-type indicate that the C-terminal tail domain of a type II keratin, including the homologous subdomain H2, is not required for filament formation. Moreover, reconstitution experiments with K8/tailless and K19, a naturally occurring tailless keratin I, show that the tail domains of type I as well as type II keratins are not an essential requirement for in vitro filament formation. Our results suggest that in vitro filament elongation does not depend on interactions between head and tail domains, although the tail domain might have a role in stabilization of intermediate filaments arising from certain keratin pairs.

scholarly journals Extremely Thermostabilizing Core Mutations in Coiled-Coil Mimetic Proteins of HIV-1 gp41 Produce Diverse Effects on Target Binding but Do Not Affect Their Inhibitory Activity

Biomolecules ◽  
2021 ◽  
Vol 11 (4) ◽  
pp. 566
Author(s):  
Mario Cano-Muñoz ◽  
Samuele Cesaro ◽  
Bertrand Morel ◽  
Julie Lucas ◽  
Christiane Moog ◽  
...  
Keyword(s):  
Inhibitory Activity ◽  
Coiled Coil ◽  
Helical Structure ◽  
Heptad Repeat ◽  
Single Chain ◽  
Cell Infection ◽  
Polar Residues ◽  
Hiv 1

A promising strategy to neutralize HIV-1 is to target the gp41 spike subunit to block membrane fusion with the cell. We previously designed a series of single-chain proteins (named covNHR) that mimic the trimeric coiled-coil structure of the gp41 N-terminal heptad repeat (NHR) region and potently inhibit HIV-1 cell infection by avidly binding the complementary C-terminal heptad repeat (CHR) region. These proteins constitute excellent tools to understand the structural and thermodynamic features of this therapeutically important interaction. Gp41, as with many coiled-coil proteins, contains in core positions of the NHR trimer several highly conserved, buried polar residues, the role of which in gp41 structure and function is unclear. Here we produced three covNHR mutants by substituting each triad of polar residues for the canonical isoleucine. The mutants preserve their helical structure and show an extremely increased thermal stability. However, increased hydrophobicity enhances their self-association. Calorimetric analyses show a marked influence of mutations on the binding thermodynamics of CHR-derived peptides. The mutations do not affect however the in vitro HIV-1 inhibitory activity of the proteins. The results support a role of buried core polar residues in maintaining structural uniqueness and promoting an energetic coupling between conformational stability and NHR–CHR binding.

scholarly journals Genetically modified rabbits as bio-producers and biomodels

2020 ◽  
Vol 224 ◽  
pp. 04034
Author(s):  
E M Koloskova ◽  
VA Ezerskiy ◽  
T P Trubitsyna ◽  
N V Belova
Keyword(s):  
Fluorescent Protein ◽  
Recombinant Dna ◽  
Genetically Modified ◽  
Biologically Active ◽  
Farm Animals ◽  
Green Fluorescent Protein Gene ◽  
Embryo Survival ◽  
Site Specific ◽  
Rabbit Gene

Genetically modified (GM) animals are necessary to solve the global problems of humanity related to nutrition and health. Rabbits, as laboratory, domestic and farm animals, occupy a special niche in research. GM rabbits are promising as bioreactors for producing biologically active (BA) proteins with milk or blood, and are in demand in Biomedicine as biomodels of diseases. To date, many GM rabbits-biomodels, producers of recombinant proteins have been created in the world using CRISPR/Cas9 technology. All-Russian Research Institute of Animal Physiology, Biochemistry and Nutrition has experience in obtaining transgenic rabbitsproducers of human BA proteins with milk by microinjecting recombinant DNA into zygote pronuclei. The possibility of site-specific modification of the rabbit whey acidic protein (WAP) gene using CRISPR/Cas9 technology is discussed. A DNA matrix containing homology arms to the WAP rabbit gene and site-specific CRISPR/Cas9 components in plasmid form were obtained. Microinjections of rabbit zygotes were performed and embryo survival was evaluated in vitro. The efficiency of using the green fluorescent protein gene under the cytomegalovirus promoter in the DNA matrix as an indicator of homologically directed repair was evaluated. This work can be useful for obtaining rabbits that produce with milk BA protein instead of WAP.

Site-specific mutagenesis of human chorionic gonadotrophin (hCG)-β subunit: influence of mutation on hCG production

1990 ◽  
Vol 5 (2) ◽  
pp. 97-102 ◽  
Author(s):  
C. Azuma ◽  
K. Miyai ◽  
F. Saji ◽  
S. Kamiura ◽  
Y. Tokugawa ◽  
...  
Keyword(s):  
In Vitro Transcription ◽  
Chorionic Gonadotrophin ◽  
Human Chorionic Gonadotrophin ◽  
Amino Acid Sequences ◽  
In Vitro Mutagenesis ◽  
Β Subunit ◽  
Site Specific ◽  
Biological Specificity ◽  
Covalently Linked

ABSTRACT The heterodimeric glycoprotein hormones, human chorionic gonadotrophin (hCG), LH, TSH and FSH, consist of two non-covalently linked subunits, the α and β subunits. The β subunit is specific for each hormone and is responsible for the biological specificity, but the β subunits of different hormones show some degree of structural homology. The CAGY (cysteine-alanine-glycine-tyrosine) region is one of the amino acid sequences that is homologous in different β subunits and is highly conserved between species. In the present study, site-specific in-vitro mutagenesis was used to change three individual nucleotides in the centre of the CAGY region of the hCG-β subunit, and the effects of these mutations on hCG production was determined by in-vitro transcription and then translation in Xenopus laevis oocytes. The results indicate that the CAGY region, particularly the glycine residue at position 36 in the β subunit, is essential for the production of hCG. This finding is consistent with previous studies showing that this region is necessary for the biological activity of human TSH.

scholarly journals In vitro reconstruction and analysis of evolutionary variation of the tomato acylsucrose metabolic network

2015 ◽  
Vol 113 (2) ◽  
pp. E239-E248 ◽  
Author(s):  
Pengxiang Fan ◽  
Abigail M. Miller ◽  
Anthony L. Schilmiller ◽  
Xiaoxiao Liu ◽  
Itai Ofner ◽  
...  
Keyword(s):  
Biosynthetic Pathway ◽  
Biochemical Analysis ◽  
In Vitro Mutagenesis ◽  
Type I ◽  
Wild Tomato Species ◽  
Tomato Species ◽  
Insect Defense ◽  
Evolutionary Variation ◽  
Ester Substrate

Plant glandular secreting trichomes are epidermal protuberances that produce structurally diverse specialized metabolites, including medically important compounds. Trichomes of many plants in the nightshade family (Solanaceae) produce O-acylsugars, and in cultivated and wild tomatoes these are mixtures of aliphatic esters of sucrose and glucose of varying structures and quantities documented to contribute to insect defense. We characterized the first two enzymes of acylsucrose biosynthesis in the cultivated tomato Solanum lycopersicum. These are type I/IV trichome-expressed BAHD acyltransferases encoded by Solyc12g006330─or S. lycopersicum acylsucrose acyltransferase 1 (Sl-ASAT1)─and Solyc04g012020 (Sl-ASAT2). These enzymes were used—in concert with two previously identified BAHD acyltransferases—to reconstruct the entire cultivated tomato acylsucrose biosynthetic pathway in vitro using sucrose and acyl-CoA substrates. Comparative genomics and biochemical analysis of ASAT enzymes were combined with in vitro mutagenesis to identify amino acids that influence CoA ester substrate specificity and contribute to differences in types of acylsucroses that accumulate in cultivated and wild tomato species. This work demonstrates the feasibility of the metabolic engineering of these insecticidal metabolites in plants and microbes.

scholarly journals Escherichia coli Extract-Based Cell-Free Expression System as an Alternative for Difficult-to-Obtain Protein Biosynthesis

2020 ◽  
Vol 21 (3) ◽  
pp. 928 ◽  
Author(s):  
Sviatlana Smolskaya ◽  
Yulia A. Logashina ◽  
Yaroslav A. Andreev
Keyword(s):  
Escherichia Coli ◽  
Recombinant Protein ◽  
Protein Expression ◽  
Recombinant Dna ◽  
Recombinant Protein Expression ◽  
Expression System ◽  
Reaction Medium ◽  
Cellular Metabolism ◽  
Heterologous Protein Expression

Before utilization in biomedical diagnosis, therapeutic treatment, and biotechnology, the diverse variety of peptides and proteins must be preliminarily purified and thoroughly characterized. The recombinant DNA technology and heterologous protein expression have helped simplify the isolation of targeted polypeptides at high purity and their structure-function examinations. Recombinant protein expression in Escherichia coli, the most-established heterologous host organism, has been widely used to produce proteins of commercial and fundamental research interests. Nonetheless, many peptides/proteins are still difficult to express due to their ability to slow down cell growth or disrupt cellular metabolism. Besides, special modifications are often required for proper folding and activity of targeted proteins. The cell-free (CF) or in vitro recombinant protein synthesis system enables the production of such difficult-to-obtain molecules since it is possible to adjust reaction medium and there is no need to support cellular metabolism and viability. Here, we describe E. coli-based CF systems, the optimization steps done toward the development of highly productive and cost-effective CF methodology, and the modification of an in vitro approach required for difficult-to-obtain protein production.

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