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PubMed2001

Molecular polymorphism of the intermediate filament protein transitin.

Transitin is an avian intermediate filament protein whose transient expression in the progenitor cells of the muscle and nerve tissues is similar to that of mammalian nestin. Both proteins contain an alpha-helical core domain flanked by a short N-terminal head and a long C-terminal extremity. However, the tail region of transitin is significantly different from that of nestin in that it harbors a unique motif containing more than 50 leucine zipper-like heptad repeats which is not found in any other intermediate filament protein. Despite the absence of introns in this region of the transitin gene, it was reported that different isoforms of the protein were produced by exclusion or inclusion of a number of repeats generated by an unusual splicing mechanism recognizing consensus 5' and 3' splice sites contained within the coding sequence of the heptad repeat domain [Napier et al. (1999) J Mol Neurosci 12:11-22]. Two monoclonal antibodies (mAbs) reacting with repeated epitopes of this motif were used to monitor transitin expression during in vitro myogenesis of the quail myogenic cell line QM7. Confocal microscopy revealed that the subcellular domains decorated with mAbs A2B11 and VAP-5 were mutually exclusive: the intermediate filament network visualized with mAb VAP-5 appeared to abut on a submembranous domain defined by mAb A2B11. When QM7 cells were induced to differentiate by switching to medium containing low serum components, an early effect was the local loss of A2B11 cortical staining at the points of cell-cell contacts. The A2B11 signal also disappeared before that of VAP-5 in newly formed myotubes. Unexpectedly, the mutually exclusive staining pattern of the mAbs could not be explained by alternative splicing since both epitopes mapped to a repeated element preceding the consensus 5' splice sites of the heptad repeat domain. An alternative explanation would be that the central repeat domain of transitin is a polymorphic structure from which different conformations exist depending on the local context. This hypothesis is strengthened by the observation that in cultured neural crest cells, the A2B11 antigen is preferentially expressed by freely migrating crest cells whose intracellular pH and calcium concentrations are different from those of non-migrating cells.

Amino Acid Sequence↗
PubMed1989

Autoimmune sequence of streptococcal M protein shared with the intermediate filament protein, vimentin.

The crossreactivity of antibodies against a renal autoimmune epitope of Streptococcus pyogenes M protein with glomerular mesangial cells was investigated. The antibodies directed against the amino acid sequence Ile-Arg-Leu-Arg of the nephritogenic type 1 M protein reacted in a fibrillar pattern with mesangial cells cultured from isolated glomeruli. In Western blots of urea-extracted mesangial proteins, the antibodies reacted with a 56-kD protein. Monoclonal and polyclonal antibodies identified the 56-kD mesangial protein as vimentin. Two synthetic peptides of human vimentin containing the sequence Arg-Leu-Arg reacted with the autoimmune antibodies raised against a streptococcal M protein peptide. These results provide evidence that the intermediate filament protein vimentin shares autoimmune epitopes with streptococcal M protein.

Amino Acid Sequence↗
PubMed1982

Cyclic AMP-modulated phosphorylation of intermediate filament proteins in cultured avian myogenic cells.

The intermediate filament proteins desmin and vimentin and the muscle tropomyosins were the major protein phosphate acceptors in 8-day-old myotubes incubated for 4 h in medium containing radiolabeled phosphate. The addition of isoproterenol or 8-bromo-cyclic AMP (BrcAMP) resulted in a two- to threefold increase in incorporation of 32PO4 into both desmin and vimentin, whereas no changes in the incorporation of 32PO4 into tropomyosin or other cellular proteins were observed. The BrcAMP- or hormonally induced increase in 32PO4 incorporation into desmin and vimentin was independent of protein synthesis and was not caused by stimulation of protein phosphate turnover. In addition, BrcAMP did not induce significant changes in the specific activity of the cellular ATP pool. These data suggest that the observed increase in 32PO4 incorporation represented an actual increase in phosphorylation of the intermediate filament proteins desmin and vimentin. Two-dimensional tryptic analysis of desmin from 8-day-old myotubes revealed five phosphopeptides of which two showed a 7- to 10-fold increase in 32PO4 incorporation in BrcAMP-treated myotubes. Four of the phosphopeptides identified in desmin labeled in vivo were also observed in desmin phosphorylated in vitro by bovine heart cAMP-dependent protein kinase. Although phosphorylation of desmin and vimentin was apparent in myogenic cells at all stages of differentiation, BrcAMP- and isoproterenol-induced increases in phosphorylation of these proteins were restricted to mature myotubes. These data strongly suggest that in vivo phosphorylation of the intermediate filament proteins desmin and vimentin is catalyzed by the cAMP-dependent protein kinases and that such phosphorylation may be regulated during muscle differentiation.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1993

Phosphorylation of a 62 kd porcine alpha-internexin, a newly identified intermediate filament protein.

A 62 kd protein was purified from the Triton-insoluble fraction of porcine brain white matter. This protein formed 10nm filaments, in vitro. The phosphorylation of the 62 kd protein by cAMP-dependent protein kinase caused electrophoretic mobility to shift to 66 kd on SDS-PAGE and a complete loss of the filament forming ability ensued. Amino acid sequences of four peptide fragments obtained from the 62 kd protein by lysylendopeptidase were identical with that of a 66 kd rat brain alpha-internexin. Amino acid analyses of the phosphopeptide fragment derived from phosphorylated porcine alpha-internexin revealed that the phosphorylation sites by cAMP-dependent protein kinase located in the amino-terminal head domain of this protein. These results strongly suggest that alpha-internexin polymerizes into 10nm filaments in vitro and that phosphorylation of the amino-terminal domain of alpha-internexin controls its polymerizability.

Amino Acid Sequence↗
PubMed1981

Two Drosophila melanogaster proteins related to intermediate filament proteins of vertebrate cells.

Monoclonal antibodies were prepared against a 46,000 mol wt major cytoplasmic protein from Drosophila melanogaster Kc cells. These antibodies reacted with the 46,000 and a 40,000 mol wt protein from Kc cells. Some antibodies showed cross-reaction with 55,000 (vimentin) and 52,000 mol wt (desmin) proteins from baby hamster kidney (BHK) cells that form intermediate sized filaments in vertebrate cells. In indirect immunofluorescence, the group of cross reacting antibodies stained a filamentous meshwork in the cytoplasm of vertebrate cells. In Kc cells the fluorescence seemed to be localized in a filamentous meshwork that became more obvious after the cells had flattened out on a surface. These cytoskeletal structures are heat-labile; the proteins in Kc or BHK cells rearrange after a brief heat shock, forming juxtanuclear cap structures.

Animals↗
PubMed1990

The development of a membrane-based screening method to detect antibodies to intermediate filament proteins.

Autoantibodies directed against the intermediate filament proteins (IF) arise in a variety of disease states. The authors have investigated the binding of the IF to solid membrane supports in a dot blot format in an attempt to develop a simple procedure to detect antibodies (ab) to IF. Commercially obtained, purified IF were utilized. These were: vimentin (VIM), cytokeratin 8 (CYK), glial fibrillary acidic protein (GFA), desmin (DES), and the neurofilament triplet proteins (68, 160, and 200 KDa, respectively designated LMW, MMW, and HMW). Murine monoclonal antibody (mAb) probes were used to detect the presence and immunoreactivity of IF. The mAb were visualized with HRP-anti-mouse conjugates using alpha-chloronaphthol/H 2O 2 as substrate. The membranes studied were nitrocellulose (NC), and two of modified nylon. Nitrocellulose provided the most reproducible binding; no advantage was found to ensue from the use of the other membranes with regard either to quantitative binding or improved capping. Among the IF studied, VIM, GFA, LMW, MMW, and HMW bound well to NC; optimal mass/dot was 1 mug. Filtered, non-fat dry milk is a better capping agent than either albumin or fetal calf serum, but interferes with ab binding to GFA. Binding of CYK and DES is weak at neutral pH. Standard densitometric techniques provide the possibility of quantitation. We conclude that dot and slot blot assays may be practical methods to detect ab to IF antigens.

Autoantibodies↗
PubMed2004

The genome of the early chordate Ciona intestinalis encodes only five cytoplasmic intermediate filament proteins including a single type I and type II keratin and a unique IF-annexin fusion protein.

We screened the recently established draft genome of the early chordate Ciona intestinalis for genes encoding cytoplasmic intermediate filament (IF) proteins. The draft of the tunicate/urochordate genome contains only the five genes (IF-A, IF-B, IF-C, IF-D and IF-F) previously established by cDNA cloning. Three of these IF proteins (IF-D, IF-C, IF-A) were shown to be orthologs of vertebrate IF subfamilies I to III while two proteins (IF-B, IF-F) seemed tunicate specific. This is now firmly established for protein IF-F since the genomic data show that it arises as a fusion protein with a C-terminal annexin domain, a feature not found before in the very large collection of metazoan IF proteins. The results also confirm the previous proposal that urochordates lack orthologs of vertebrate type IV IF proteins. We discuss the striking increase of IF complexity from 5 tunicate to 65 human genes during chordate evolution. Thus the tunicate has a single keratin pair, which is expressed in the epidermis, while the human genome has at least 25 genes each for keratins I and keratins II. Finally there are four normal Ciona annexin genes in addition to the gene encoding the IF-annexin fusion proteins (IF-F).

Amino Acid Sequence↗
PubMed1992

p13suc1 suppresses the catalytic function of p34cdc2 kinase for intermediate filament proteins, in vitro.

The regulation of p34cdc2 kinase activity controls the entry into and exit from mitosis. Although genetic and biochemical evidence suggested close interactions between cyclins, p13suc1 and p34cdc2 kinase, the roles of p13suc1 on p34cdc2 kinase functions remain unclear. To examine the effects of p13suc1 on p34cdc2 kinase function we developed a simple purification procedure for p34cdc2 kinase, unassociated with p13suc1. The key to the purification procedures we used was buffer containing 0.5 M NaCl and 50% ethylene glycol, as a specific elutant of p34cdc2 kinase from p13suc1-Sepharose. This purified p34cdc2 kinase stoichiometrically phosphorylated vimentin and desmin. Exogenous p13suc1 suppressed the phosphorylation of these filament proteins by the kinase and prevented disassembly, although histone H1 phosphorylation was not affected. Peptide mapping analysis showed a similar extent of inhibition by p13suc1 for all five phosphorylation sites by p34cdc2 kinase of vimentin and desmin, hence these p13suc1-induced inhibitions are probably not site-specific. It thus appears that p13suc1 has a selective effect on the catalytic activity of p34cdc2 kinase for these filament proteins.

Amino Acid Sequence↗
PubMed1999

Analysis of the roles of the head domains of type IV rat neuronal intermediate filament proteins in filament assembly using domain-swapped chimeric proteins.

Type IV neuronal intermediate filament proteins consist of alpha-internexin, which can self-assemble into filaments and the neurofilament triplet proteins, which are obligate heteropolymers, at least in rodents. These IF proteins therefore provide good systems for elucidating the mechanism of intermediate filament assembly. To analyze the roles of the head domains of these proteins in contributing to their differential assembly properties, we generated chimeric proteins by swapping the head domains between rat alpha-internexin and either rat NF-L or NF-M and examined their assembly properties in transfected cells that lack their own cytoplasmic intermediate filament network. Lalphaalpha and Malphaalpha, the chimeric proteins generated by replacing the head domain of alpha-internexin with those of NF-L and NF-M, respectively, were unable to self-assemble into filaments. In contrast, alphaLL, a chimeric NF-L protein generated by replacing the head domain of NF-L with that of alpha-internexin, was able to self-assemble into filaments, whereas MLL, a chimeric NF-L protein containing the NF-M head domain, was unable to do so. These results demonstrate that the alpha-internexin head domain is essential for alpha-internexin's ability to self-assemble. While coassembly of Lalphaalpha with NF-M and coassembly of Malphaalpha with NF-L resulted in formation of filaments, coassembly of Lalphaalpha with NF-L and coassembly of Malphaalpha with NF-M yielded punctate patterns. These coassembly results show that heteropolymeric filament formation requires that one partner has the NF-L head domain and the other partner has the NF-M head domain. Thus, the head domains of rat NF-L and NF-M play important roles in determining the obligate heteropolymeric nature of filament formation. The data obtained from these self-assembly and coassembly studies provide some new insights into the mechanism of intermediate filament assembly.

Amino Acid Sequence↗
PubMed1999

Intermediate filament proteins in developing human arteries.

The distribution of intermediate filament proteins in adult human blood vessels and in human fetal elastic arteries is relatively well-known. However, the distribution of these proteins in the course from neonate to adult has not been established. In this investigation, human postnatal arteries were studied with immunohistochemistry, using antibodies targeted on the intermediate filament proteins desmin, vimentin and cytokeratins 8, 18 and 19. Vimentin was present in most smooth muscle cells in all vessels and at all ages. The proportions of desmin-expressing cells increased in the elastic arteries during the first year of life and was higher in the pulmonary trunk than in the aorta. In the muscular arteries, the proportion of desmin-labelled cells increased in the coronary and the deep femoral arteries, but remained constant in the renal and the cerebral arteries. Cytokeratins were detected in the pulmonary trunk earlier than in the aorta. Cytokeratins were present throughout the wall of the ductus arteriosus, but desmin was present only in some cells. Thus, there are postnatal changes in the distribution of intermediate filament proteins in the elastic arteries and in some muscular arteries, whereas the intermediate filament pattern remains unchanged in other muscular arteries.

Adolescent↗
PubMed2004

Desmin: molecular interactions and putative functions of the muscle intermediate filament protein.

Desmin is the intermediate filament (IF) protein occurring exclusively in muscle and endothelial cells. There are other IF proteins in muscle such as nestin, peripherin, and vimentin, besides the ubiquitous lamins, but they are not unique to muscle. Desmin was purified in 1977, the desmin gene was characterized in 1989, and knock-out animals were generated in 1996. Several isoforms have been described. Desmin IFs are present throughout smooth, cardiac and skeletal muscle cells, but can be more concentrated in some particular structures, such as dense bodies, around the nuclei, around the Z-line or in costameres. Desmin is up-regulated in muscle-derived cellular adaptations, including conductive fibers in the heart, electric organs, some myopathies, and experimental treatments with drugs that induce muscle degeneration, like phorbol esters. Many molecules have been reported to associate with desmin, such as other IF proteins (including members of the membrane dystroglycan complex), nebulin, the actin and tubulin binding protein plectin, the molecular motor dynein, the gene regulatory protein MyoD, DNA, the chaperone alphaB-crystallin, and proteases such as calpain and caspase. Desmin has an important medical role, since it is used as a marker of tumors' origin. More recently, several myopathies have been described, with accumulation of desmin deposits. Yet, after almost 30 years since its identification, the function of desmin is still unclear. Suggested functions include myofibrillogenesis, mechanical support for the muscle, mitochondrial localization, gene expression regulation, and intracellular signaling. This review focuses on the biochemical interactions of desmin, with a discussion of its putative functions.

Animals↗
PubMed1998

Primary sequence, secondary structure, gene structure, and assembly properties suggests that the lens-specific cytoskeletal protein filensin represents a novel class of intermediate filament protein.

The ocular lens fiber cell assembles a novel cytoskeletal element, the Beaded Filament, from CP49 and filensin, two proteins expressed only in the differentiated lens fiber cell. We report the primary sequence, secondary structural analysis, gene structure and Yeast Two Hybrid interaction data for human filensin, and develop a consensus model of filensin from the human and previously reported bovine and chicken filensin sequences. This consensus model, combined with gene structure and Yeast Two Hybrid studies establish that filensin is a member of the Intermediate Filament family of proteins. Specifically, filensin exhibits (1) divergence at amino acid sequence motifs otherwise highly conserved among intermediate filament proteins, (2) a loss of 29 amino acids from the central rod domain which is unique among cytoplasmic intermediate filament proteins, (3) an absence of sequence identity with any existing class of intermediate filament protein, (4) a gene structure unique among intermediate filament family, (5) an inability to dimerize with representatives of Type I, II, and III intermediate filament proteins. Thus, at each level of analysis, we find that filensin is similar to the consensus model of intermediate filament proteins, supporting our conclusion that filensin's relatedness to the IF family is not the consequence of convergent evolution. However, filensin also shows unique or extreme distinctions from the consensus intermediate filament protein at each level of analysis, indicating that filensin constitutes a novel class of IF protein. Some of filensin's unique features are incompatible with current models of IF assembly. Analysis of filensin gene structure suggests that the 29 amino acid reduction in the central rod domain was not the result of a single splice site mutation, the mechanism suggested for the transition between nuclear lamins and cytoplasmic intermediate filament proteins.

Amino Acid Sequence↗
PubMed1989

Evolution of homologous domains of cytoplasmic intermediate filament proteins and lamins.

The earliest gene duplications in the evolution of the intermediate filament proteins created the ancestors of acidic keratins, basic keratins, nonepithelial intermediate filament proteins, and lamins. Biochemistry and function of cytoplasmic intermediate filaments differ greatly from those of lamins. Cytoplasmic intermediate filament proteins have a different cellular location than lamins, form different types of supramolecular structures, and are missing a protein segment found in lamins; but the data presented here indicate that the cytoplasmic intermediate filaments do not have a common ancestor separate from the ancestor of lamins. In the non-epithelial intermediate filament branch, the ancestor of neurofilament proteins and the common ancestor of desmin, vimentin, and glial fibrillary acidic protein (GFAP) diverged first. By evolutionary criteria, the intermediate filament protein recently discovered in neuronal cells does not belong to the neurofilament family but is more closely related to desmin, vimentin, and GFAP. Sequences of different sub-domains yield different evolutionary trees, possibly indicating existence of sub-domain-specific functions.

Amino Acid Sequence↗
PubMed1988

Immunocytochemical identification of non-neuronal intermediate filament proteins in the developing Xenopus laevis nervous system.

Intermediate filament proteins in the postmetamorphic Xenopus laevis nervous system were identified by their crossreactivities on Western blots with a pan-specific intermediate filament antibody (anti-IFA). These intermediate filament protein bands on Western blots were characterized as 3 cytokeratin-like proteins (49, 55, and 58 kDa), one vimentin-like protein (53 kDa), two distinct glial fibrillary acidic protein (GFAP)-like proteins (60 and 67 kDa), and 3 neurofilament proteins (73, 175, and 200 kDa) by evaluation of their crossreactivities with specific antibodies directed against the mammalian forms of these proteins. This panel of antibodies to mammalian proteins, and two additional antibodies directed against a Xenopus GFAP-like protein and a Xenopus neurofilament (NF-M) protein, were used in immunocytochemical studies to determine the developmental expression of these proteins in the Xenopus nervous system. The first antigen to be detected during development was cytokeratin immunoreactivity, which was located in the inner lining of the embryonic neural tube as early as stage 19, and which in immunocytochemical studies in postmetamorphic frogs was abundant in meninges and processes forming the ventricular lining of the ependymal zone. Vimentin immunoreactivity was found in numerous neuroepithelial cell processes in the rhombencephalon and anterior spinal cord by stage 22, in the prosencephalon by stage 33/34, and in the retina by stage 29/30. In the postmetamorphic frog, vimentin immunoreactivity was found to be abundant in radial processes throughout the brain and spinal cord. NF-M protein immunoreactivity was first detected in neurons in the developing neural tube between stages 22 and 24, in the retina by stages 29/30, and continued to increase throughout development. GFAP-like immunoreactivity was detected very early in radial cells in the neural tube (stage 24), and by stage 42 was found throughout the nervous system. This early appearance of GFAP-like immunoreactivity implies that the onset of glial cell differentiation is a relatively early event in Xenopus.

Animals↗
PubMed1988

Amino acid sequences and homopolymer-forming ability of the intermediate filament proteins from an invertebrate epithelium.

Intermediate filaments (IF) isolated from the oesophagus epithelium of the snail Helix pomatia contain two polypeptides of mol. wt 66,000 (A) and 52,000 (B), which we have now characterized by in vitro self-assembly studies and by protein sequences. A and B can each form morphologically normal IF and share extended regions of sequence identity. All A-specific sequences seem to locate to an extension of the carboxyl-terminal domain. Although the Helix protein(s) reveal the IF-consensus sequences at the ends of the coiled-coil, the remainder of the rod domain shows conservation of sequence principles rather than extended homology, when compared with any subtype of vertebrate IF proteins. Interestingly, the Helix proteins have the longer coil 1b domain found in nuclear lamins and not in cytoplasmic IF proteins of vertebrates. They lack, however, the karyophilic signal sequence typical for lamins. Obvious implications for IF evolution and structure are discussed.

Amino Acid Sequence↗
PubMed1998

Specific interaction of HTLV tax protein and a human type IV neuronal intermediate filament protein.

The human T-cell leukemia virus (HTLV) is associated with adult T cell leukemia and neurological disorders (TSP/HAM). The HTLV transcriptional transactivator, Tax, is known to exert its effect through protein-protein interaction with several transcription factors that activate genes in T cell proliferation. The pathogenic mechanism in the CNS is less defined. Using the yeast two-hybrid system, we have identified a specific Tax-binding protein as the neuronal specific intermediate filament protein, alpha-internexin. Tax binds to the domain corresponding to the rod region of alpha-internexin, which is essential for neurofilament assembly. The Tax domains involved in binding are separable from those involved in transactivation. TxBP-1/alpha-internexin and Tax are expressed in the cytoplasm and nucleus, respectively, when expressed alone, but in coexpressing cells, colocalization of both proteins was observed in a perinuclear, punctate distribution. This in vivo interaction also resulted in a dramatic reduction in Tax transactivation and the network formation by alpha-internexin. The specific interaction of Tax and a neuronal specific intermediate filament protein may provide a clue to the pathogenesis of TSP/HAM.

Amino Acid Sequence↗
PubMed1982

Studies on the biosynthesis of intermediate filament proteins in the rat CNS.

The biosynthesis of brain intermediate filament proteins [neurofilament proteins and glial fibrillary acidic protein (GFA)] was studied with cell-free systems containing either rat spinal cord polysomes (free polysomes or rough microsomes) and rabbit reticulocyte factors or wheat germ homogenate containing spinal cord messenger RNA. The products of translation were isolated by immunoaffinity chromatography and then analyzed by two-dimensional gel electrophoresis (2DGE) followed by fluorography. The free polysome population was found to synthesize two neurofilament proteins (MW 145K, pI 5.4, and MW 70K, pI 5.3) and three isomers of GFA (alpha, beta, and gamma) that differ in isoelectric point. Wheat germ homogenate containing messenger RNA extracted from free cord polysomes synthesized two proteins that comigrated with neurofilament protein standards at 145K 5.4 and 70K 5.3; these proteins were partially purified by neurofilament affinity chromatography. The wheat germ system also synthesized the alpha, beta, and gamma isomers of GFA as characterized by immunoaffinity chromatographic purification and comigration with standards in 2DGE analysis. Our data are consistent with the conclusion that synthesis of neurofilament proteins requires multiple messenger RNAs. Also, synthesis of intermediate filament proteins occurs in the free polysome population; detectable amounts of these proteins were not synthesized by the rough microsomes.

Animals↗