scholarly journals An unusual intermediate filament subunit from the cytoskeletal biopolymer released extracellularly into seawater by the primitive hagfish (Eptatretus stouti)

1994 ◽  
Vol 107 (11) ◽  
pp. 3133-3144 ◽  
Author(s):  
E.A. Koch ◽  
R.H. Spitzer ◽  
R.B. Pithawalla ◽  
D.A. Parry
Keyword(s):  
Intermediate Filament ◽  
Coiled Coil ◽  
Total Content ◽  
Data Bank ◽  
Peripheral Region ◽  
Amino Acid Sequences ◽  
Type I ◽  
10 Nm Filaments ◽  
Terminal Domains

Each slime gland thread cell from the primitive Pacific hagfish (Eptatretus stouti) contains a massive, conical, intermediate filament (IF)-rich biopolymer (‘thread,’ approximately 60 cm length, approximately 3 microns width). In view of the unusual ultrastructure of the thread, its extracellular role in modulation of the viscoelastic properties of mucus, and the ancient lineage of this primitive vertebrate, we report the nucleotide and deduced amino acid sequences of one major thread IF subunit, alpha (pI 7.5), which is coexpressed with a second polypeptide, gamma (pI 5.3). These two polypeptides coassemble in vitro into approximately 10 nm filaments. The alpha-thread chain, a 66.6 kDa polypeptide, has an unusual central rod domain containing 318 residues flanked by N- and C-terminal domains of 192 and 133 residues, respectively. Each peripheral region exhibits some epidermal keratin-like features including peptide repeats and a high total content of glycine and serine residues. The terminal domains, however, lack the H1 and H2 subdomains characteristic of known keratins. Moreover, when the central rod is aligned either in relation to established homology profiles (J. F. Conway and D. A. D. Parry (1988) Int. J. Biol. Macromol. 10, 79–98) of other IF subunits (types I-V, nestin, non-neuronal invertebrate), or by computer-based homology searches of the GenBank/EMBL Data Bank, a low identity (< 30%) is evident, with no preferred identity to keratins or other known IF types. Although the central rod of 318 residues consists of the canonical apolar heptad repeats interspersed with three linker regions, a discontinuity in phasing of the heptad substructure in rod 2B, and conserved sequences at either end of the rod domain, other collective characteristics are atypical: overall high threonine content (13.2% vs 2.3-5.4% for other IFs), high threonine content in rod 1B (18.8% vs 1–6%), five Thr-Thr repeats in coiled coil segments, L12 of length greater than in keratins, substitution of phenylalanine for a highly conserved glutamate in the sixth position of L2, and a glycine-proline sequence in segment 2B. Possibly as a result of the high threonine content, the percentage of both acidic and basic residues in most helical subdomains is reduced relative to type I and II chains. Fast Fourier transform analyses show that only the acidic residues in segment 1B and basic residues in segment 2 have near typical IF periods.(ABSTRACT TRUNCATED AT 400 WORDS)

scholarly journals Revealing biophysical properties of KfrA-type proteins as a novel class of cytoskeletal, coiled-coil plasmid-encoded proteins

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
M. Adamczyk ◽  
E. Lewicka ◽  
R. Szatkowska ◽  
H. Nieznanska ◽  
J. Ludwiczak ◽  
...  
Keyword(s):  
Dna Binding ◽  
Host Range ◽  
Coiled Coil ◽  
Amino Acid Sequences ◽  
Broad Host Range ◽  
Biophysical Properties ◽  
Dna Binding Sites ◽  
In Silico Studies ◽  
Wide Range

Abstract Background DNA binding KfrA-type proteins of broad-host-range bacterial plasmids belonging to IncP-1 and IncU incompatibility groups are characterized by globular N-terminal head domains and long alpha-helical coiled-coil tails. They have been shown to act as transcriptional auto-regulators. Results This study was focused on two members of the growing family of KfrA-type proteins encoded by the broad-host-range plasmids, R751 of IncP-1β and RA3 of IncU groups. Comparative in vitro and in silico studies on KfrAR751 and KfrARA3 confirmed their similar biophysical properties despite low conservation of the amino acid sequences. They form a wide range of oligomeric forms in vitro and, in the presence of their cognate DNA binding sites, they polymerize into the higher order filaments visualized as “threads” by negative staining electron microscopy. The studies revealed also temperature-dependent changes in the coiled-coil segment of KfrA proteins that is involved in the stabilization of dimers required for DNA interactions. Conclusion KfrAR751 and KfrARA3 are structural homologues. We postulate that KfrA type proteins have moonlighting activity. They not only act as transcriptional auto-regulators but form cytoskeletal structures, which might facilitate plasmid DNA delivery and positioning in the cells before cell division, involving thermal energy.

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 Developmentally regulated cytokeratin gene in Xenopus laevis.

1985 ◽  
Vol 5 (10) ◽  
pp. 2575-2581 ◽  
Author(s):  
J A Winkles ◽  
T D Sargent ◽  
D A Parry ◽  
E Jonas ◽  
I B Dawid
Keyword(s):  
Amino Acid ◽  
Xenopus Laevis ◽  
Intermediate Filament ◽  
Type I ◽  
Developmentally Regulated ◽  
Amino Acid Homology ◽  
Intermediate Filament Proteins ◽  
Functional Roles ◽  
Terminal Domain ◽  
Terminal Domains

We have determined the sequence of cloned cDNAs derived from a 1,665-nucleotide mRNA which transiently accumulates during Xenopus laevis embryogenesis. Computer analysis of the deduced amino acid sequence revealed that this mRNA encodes a 47-kilodalton type I intermediate filament subunit, i.e., a cytokeratin. As is common to all intermediate filament subunits so far examined, the predicted polypeptide, named XK70, contains N- and C-terminal domains flanking a central alpha-helical rod domain. The overall amino acid homology between XK70 and a human 50-kilodalton type I keratin is 47%; homology within the alpha-helical domain is 57%. The N-terminal domain, which is not completely contained in our cDNAs, is basic, contains 42% serine plus alanine, and includes five copies of a six-amino-acid repeating unit. The C-terminal domain has a high alpha-helical content and contains a region with sequence homology to the C-terminal domains of other type I and type III intermediate filament proteins. We suggest that different keratin filament subtypes may have different functional roles during amphibian oogenesis and embryogenesis.

scholarly journals The amino acid sequence of component 7c, a type II intermediate-filament protein from wool

1989 ◽  
Vol 261 (3) ◽  
pp. 1015-1022 ◽  
Author(s):  
L G Sparrow ◽  
C P Robinson ◽  
D T W McMahon ◽  
M R Rubira
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Intermediate Filament ◽  
Coiled Coil ◽  
British Library ◽  
Intermediate Filament Protein ◽  
Type I ◽  
Type Ii ◽  
Blocking Group ◽  
Intermediate Filament Proteins

Component 7c is one of the four homologous type II intermediate-filament proteins that, by association with the complementary type I proteins, form the microfibrils or intermediate filaments in wool. Component 7c was isolated as the S-carboxymethyl derivative from Merino wool and its amino acid sequence was determined by manual and automatic sequencing of peptides produced by chemical and enzymic cleavage reactions. It is an N-terminally blocked molecule of 491 residues and Mr (not including the blocking group) of 55,600; the nature of the blocking group has not been determined. The predicted secondary structure shows that component 7c conforms to the now accepted pattern for intermediate-filament proteins in having a central rod-like region of approximately 310 residues of coiled-coil alpha-helix flanked by non-helical N-and C-terminal regions. The central region is divided by three non-coiled-coil linking segments into four helical segments 1A, 1B, 2A and 2B. The N-and C-terminal non-helical segments are 109 and 71 residues respectively and are rich in cysteine. Details of procedures use in determining the sequence of component 7c have been deposited as a Supplementary Publication SUP 50152 (65 pages) at the British Library Document Supply Centre, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1989) 257,5. The information comprises: (1) details of chemical and enzymic methods used for cleavage of component 7c, peptides CN1, CN2 and CN3, and various other peptides, (2) details of the procedures used for the fractionation and purification of peptides from (1), including Figures showing the elution profiles from the chromatographic steps used, (3) details of methods used to determine the C-terminal sequence of peptide CN3, and (4) detailed evidence to justify a number of corrections to the previously published sequence.

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 Amino acid sequences of α-helical segments from S-carboxymethylkerateine-A. Complete sequence of a type-II segment

1978 ◽  
Vol 173 (2) ◽  
pp. 365-371 ◽  
Author(s):  
W G Crewther ◽  
A S Inglis ◽  
N M McKern
Keyword(s):  
Amino Acid ◽  
Coiled Coil ◽  
Complete Sequence ◽  
Amino Acid Sequences ◽  
Helical Axis ◽  
Type I ◽  
Type Ii ◽  
Coil Structure ◽  
Aqueous Urea ◽  
Alpha Keratin

1. The helical fragments obtained by partial chymotryptic digestion of S-carboxymethylkeratine-A, the low-sulphur fraction from wool, were fractionated into type-I and type-II helical segments in aqueous urea under conditions limiting carbamoylation. 2. The amino acid sequence of a 109-residue type-II segment was completed by using the sequenator. 3. When the data were incorporated into a helical model of 3.6 residues per turn the hydrophobic residues generated a band aligned at a slight angle to the helical axis. This result is in accord with the postulated coiled-coil structure of the crystalline regions of alpha-keratin.

Dose-dependent linkage, assembly inhibition and disassembly of vimentin and cytokeratin 5/14 filaments through plectin's intermediate filament-binding domain

2000 ◽  
Vol 113 (3) ◽  
pp. 483-491 ◽  
Author(s):  
F.A. Steinbock ◽  
B. Nikolic ◽  
P.A. Coulombe ◽  
E. Fuchs ◽  
P. Traub ◽  
...  
Keyword(s):  
Intermediate Filament ◽  
Ectopic Expression ◽  
Type I ◽  
Dependent Manner ◽  
Tail Region ◽  
Concentration Dependent Manner ◽  
Native Proteins ◽  
Dose Dependent

Plectin, the largest and most versatile member of the cytolinker/plakin family of proteins characterized to date, has a tripartite structure comprising a central 200 nm-long (α)-helical rod domain flanked by large globular domains. The C-terminal domain comprises a short tail region preceded by six highly conserved repeats (each 28–39 kDa), one of which (repeat 5) contains plectin's intermediate filament (IF)-binding site. We used recombinant and native proteins to assess the effects of plectin repeat 5-binding to IF proteins of different types. Quantitative Eu(3+)-based overlay assays showed that plectin's repeat 5 domain bound to type III IF proteins (vimentin) with preference over type I and II cytokeratins 5 and 14. The ability of both types of IF proteins to self-assemble into filaments in vitro was impaired by plectin's repeat 5 domain in a concentration-dependent manner, as revealed by negative staining and rotary shadowing electron microscopy. This effect was much more pronounced in the case of vimentin compared to cytokeratins 5/14. Preassembled filaments of both types became more and more crosslinked upon incubation with increasing concentrations of plectin repeat 5. However, at high proportions of plectin to IF proteins, disassembly of filaments occurred. Again, vimentin filaments proved considerably more sensitive towards disassembly than those composed of cytokeratins 5 and 14. In general, IFs formed from recombinant proteins were found to be slightly more responsive towards plectin influences than their native counterparts. A dose-dependent plectin-inflicted collapse and putative disruption of IFs was also observed in vivo after ectopic expression of vimentin and plectin's repeat 5 domain in cotransfected vimentin-deficient SW13 (vim(-)) cells. Our results suggest an involvement of plectin not only in crosslinking and stabilization of cytoskeletal IF networks, but also in regulation of their dynamics.

scholarly journals Deletions in epidermal keratins leading to alterations in filament organization in vivo and in intermediate filament assembly in vitro.

1990 ◽  
Vol 111 (6) ◽  
pp. 3049-3064 ◽  
Author(s):  
P A Coulombe ◽  
Y M Chan ◽  
K Albers ◽  
E Fuchs
Keyword(s):  
Intermediate Filament ◽  
Intermediate Filament Proteins ◽  
Amino Terminal ◽  
Filament Assembly ◽  
Keratin Filaments ◽  
Keratin Filament ◽  
Filament Networks ◽  
10 Nm Filaments

To investigate the sequences important for assembly of keratins into 10-nm filaments, we used a combined approach of (a) transfection of mutant keratin cDNAs into epithelial cells in vivo, and (b) in vitro assembly of mutant and wild-type keratins. Keratin K14 mutants missing the nonhelical carboxy- and amino-terminal domains not only integrated without perturbation into endogenous keratin filament networks in vivo, but they also formed 10-nm filaments with K5 in vitro. Surprisingly, keratin mutants missing the highly conserved L L E G E sequence, common to all intermediate filament proteins and found at the carboxy end of the alpha-helical rod domain, also assembled into filaments with only a somewhat reduced efficiency. Even a carboxy K14 mutant missing approximately 10% of the rod assembled into filaments, although in this case filaments aggregated significantly. Despite the ability of these mutants to form filaments in vitro, they often perturbed keratin filament organization in vivo. In contrast, small truncations in the amino-terminal end of the rod domain more severely disrupted the filament assembly process in vitro as well as in vivo, and in particular restricted elongation. For both carboxy and amino rod deletions, the more extensive the deletion, the more severe the phenotype. Surprisingly, while elongation could be almost quantitatively blocked with large mutations, tetramer formation and higher ordered lateral interactions still occurred. Collectively, our in vitro data (a) provide a molecular basis for the dominance of our mutants in vivo, (b) offer new insights as to why different mutants may generate different phenotypes in vivo, and (c) delineate the limit sequences necessary for K14 to both incorporate properly into a preexisting keratin filament network in vivo and assemble efficiently into 10-nm keratin filaments in vitro.

scholarly journals Amino acid sequences of α-helical segments from S-carbosymethylkerateine-A. Complete sequence of a type-I segment

1978 ◽  
Vol 173 (2) ◽  
pp. 373-385 ◽  
Author(s):  
K H Gough ◽  
A S Inglis ◽  
W G Crewther
Keyword(s):  
Amino Acid ◽  
Sequence Data ◽  
Coiled Coil ◽  
Alpha Helix ◽  
Protein S ◽  
Amino Acid Sequences ◽  
Type I ◽  
Type Ii ◽  
Sequencing Data ◽  
Helical Segment

The amino acid sequence of a type-I helical segment from the low-sulphur protein (S-carboxymethylkerateine-A) of wool was determined by combining automatic and manual-sequencing data. Whereas in the type-II helical segment most of the cationic groups occur in pairs, 11 of the 22 anionic residues in the sequence of the type-I segment were situated next to a second anionic residue. This suggests possible interactions between type-I and type-II helical segments in alpha-keratin. As observed with the sequence of a type-II helical segment a model constructed on 3.6 residues per turn of helix shows a line of hydrophobic residues along the helix, thereby supporting the physicochemical evidence that the molecule is predominantly helical and forms part of a coiled-coil structure. Examination of the sequence data by predictive methods indicates the possibilty of extensive sections of alpha-helix interspersed with discontinuities. The molecule contains a number of regions with peptide sequences identical with those found by other workers after enzymic digestion of fractions from oxidized wool.

scholarly journals Self-organization of keratin intermediate filaments into cross-linked networks

2009 ◽  
Vol 186 (3) ◽  
pp. 409-421 ◽  
Author(s):  
Chang-Hun Lee ◽  
Pierre A. Coulombe
Keyword(s):  
Epithelial Cells ◽  
Self Organization ◽  
Live Cells ◽  
Type I ◽  
Filamentous Actin ◽  
Basal Progenitor ◽  
Filament Bundles ◽  
10 Nm Filaments

Keratins, the largest subgroup of intermediate filament (IF) proteins, form a network of 10-nm filaments built from type I/II heterodimers in epithelial cells. A major function of keratin IFs is to protect epithelial cells from mechanical stress. Like filamentous actin, keratin IFs must be cross-linked in vitro to achieve the high level of mechanical resilience characteristic of live cells. Keratins 5 and 14 (K5 and K14), the main pairing occurring in the basal progenitor layer of epidermis and related epithelia, can readily self-organize into large filament bundles in vitro and in vivo. Here, we show that filament self-organization is mediated by multivalent interactions involving distinct regions in K5 and K14 proteins. Self-organization is determined independently of polymerization into 10-nm filaments, but involves specific type I–type II keratin complementarity. We propose that self-organization is a key determinant of the structural support function of keratin IFs in vivo.

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