Separation of wool intermediate filament protein and intermediate filament-associated protein on a Sepharose column.
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Amino Acids↗
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Results for “Intermediate Filament Proteins”
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Phosphorylation of glial fibrillary acidic protein and vimentin by cytoskeletal-associated intermediate filament protein kinase activity in astrocytes.
These studies describe a cytoskeletal-associated protein kinase activity in astrocytes that phosphorylated the intermediate filament proteins glial fibrillary acidic protein (GFAP) and vimentin and that appeared to be distinct from protein kinase C (PK-C) and the cyclic AMP-dependent protein kinase (PK-A). The cytoskeletal-associated kinase activity phosphorylated intermediate filament proteins in the presence of 10 mM MgCl2 and produced an even greater increase in 32P incorporation into these proteins in the presence of calcium/calmodulin. Tryptic peptide mapping of phosphorylated intermediate filament proteins showed that the intermediate filament protein kinase activity produced unique phosphopeptide maps, in both the presence and the absence of calcium/calmodulin, as compared to that of PK-C and PK-A, although there were some common sites of phosphorylation among the kinases. In addition, it was determined that the intermediate filament protein kinase activity phosphorylated both serine and threonine residues of the intermediate filament proteins, vimentin and GFAP. However, the relative proportion of serine and threonine residues phosphorylated varied depending on the presence or absence of calcium/calmodulin. The magnesium-dependent activity produced the highest proportion of threonine phosphorylation, suggesting that the calcium/calmodulin-dependent kinase activity acts mainly at serine residues. PK-A and PK-C phosphorylated mainly serine residues. Also, the intermediate filament protein kinase activity phosphorylated both the N-and the C-terminal domains of vimentin and the N-terminal domain of GFAP. In contrast, both PK-C and PK-A are known to phosphorylate the N-terminal domains of both proteins.(ABSTRACT TRUNCATED AT 250 WORDS)
Amino Acids↗
Human immunodeficiency virus type 1 protease cleaves the intermediate filament proteins vimentin, desmin, and glial fibrillary acidic protein.
The intermediate filament proteins vimentin, desmin, and glial fibrillary acidic protein are cleaved in vitro by human immunodeficiency virus type 1 protease (HIV-1 PR). Microsequencing showed that HIV-1 PR cleaved both human and murine vimentin between leucine-422 and arginine-423 within the sequence between positions 418 and 427, Ser-Ser-Leu-Asn-Leu/Arg-Glu-Thr-Asn-Leu (SSLNL/RETNL). Minor cleavages at other sites were also observed. Heat-denatured vimentin was cleaved by HIV-1 PR less efficiently than native vimentin. A decapeptide containing the sequence SSLN-LRETNL was also cleaved in vitro by HIV-1 PR as predicted. The presence of a charged residue (arginine) at the primary cleavage site distinguishes this from other known naturally occurring cleavage sites. Microinjection of HIV-1 PR into cultured human fibroblasts resulted in a 9-fold increase in the percentage of cells with an altered and abnormal distribution of vimentin intermediate filaments. Most commonly, the intermediate filaments collapsed into a clump with a juxtanuclear localization. These results support the possibility that intermediate filament proteins may serve as substrates within HIV-1-infected cells.
Amino Acid Sequence↗
Cytoplasmic intermediate filament proteins of invertebrates are closer to nuclear lamins than are vertebrate intermediate filament proteins; sequence characterization of two muscle proteins of a nematode.
The giant body muscle cells of the nematode Ascaris lumbricoides show a complex three dimensional array of intermediate filaments (IFs). They contain two proteins, A (71 kd) and B (63 kd), which we now show are able to form homopolymeric filaments in vitro. The complete amino acid sequence of B and 80% of A have been determined. A and B are two homologous proteins with a 55% sequence identity over the rod and tail domains. Sequence comparisons with the only other invertebrate IF protein currently known (Helix pomatia) and with vertebrate IF proteins show that along the coiled-coil rod domain, sequence principles rather than actual sequences are conserved in evolution. Noticeable exceptions are the consensus sequences at the ends of the rod, which probably play a direct role in IF assembly. Like the Helix IF protein the nematode proteins have six extra heptads in the coil 1b segment. These are characteristic of nuclear lamins from vertebrates and invertebrates and are not found in vertebrate IF proteins. Unexpectedly the enhanced homology between lamins and invertebrate IF proteins continues in the tail domains, which in vertebrate IF proteins totally diverge. The sequence alignment necessitates the introduction of a 15 residue deletion in the tail domain of all three invertebrate IF proteins. Its location coincides with the position of the karyophilic signal sequence, which dictates nuclear entry of the lamins. The results provide the first molecular support for the speculation that nuclear lamins and cytoplasmic IF proteins arose in eukaryotic evolution from a common lamin-like predecessor.
Amino Acid Sequence↗
Microinjection of intermediate filament proteins into living cells with and without preexisting intermediate filament network.
Human cells grown in monolayer culture were microinjected with intermediate filament subunit proteins. In fibroblasts with a preexisting vimentin network, injected porcine glial fibrillary acidic protein (GFAP) co-localized with the vimentin network within 24 hours. Phosphorylated GFAP variants were found to become dephosphorylated concomitantly with their incorporation into filamentous structures. After microinjection of either porcine GFAP or murine vimentin into human carcinoma cells lacking cytoplasmic intermediate filaments, we observed that different types of filament networks developed. Whereas vimentin was incorporated into short filaments immediately after injection, GFAP was found to aggregate into rodlike structures. This may indicate a differential filament forming ability of these intermediate filament proteins in vivo.
Cell Line↗
Salt-stable interaction of the amino-terminal head region of vimentin with the alpha-helical rod domain of cytoplasmic intermediate filament proteins and its relevance to protofilament structure and filament formation and stability.
Previous studies have shown that the non-alpha-helical, amino-terminal head region of vimentin is essential for the formation and stability of vimentin intermediate filaments (IFs). In order to specify its target site on companion protein subunits, it was cut off from vimentin at amino acid position 96 with lysine-specific endoproteinase and allowed to react with intact vimentin and other IF proteins. In solution of high salt concentration (500 mM KCl), the isolated polypeptide (vim NT) showed a high affinity for all cytoplasmic IF proteins tested, but not for nuclear lamins. Employing limited digestion of the IF proteins with different proteinases, the binding site was shown to reside in their alpha-helical rod domains. Other polypeptides possessing alpha-helical regions with the potential to form coiled-coil structures like tropomyosin and myosin subfragment 2 did not react with vim NT. The binding to IF proteins was strongly inhibited by phosphorylation of vim NT and totally abolished in the presence of 200 mM arginine hydrochloride, whereas the same concentration of lysine hydrochloride was ineffective. Limited chymotryptic digestion of vim NT produced polypeptides that were unable to react with the alpha-helical region of vimentin at high salt concentration. Consistent with these observations, vim NT strongly inhibited filament formation in vitro from protofilamentous vimentin. A 14-mer oligopeptide comprising the amino acids 3 to 16 of the amino terminus also inhibited filament formation, though to a lesser extent. Conversely, vim NT and, with a lower efficiency, the 14-mer oligopeptide also severely affected the structure of preformed vimentin filaments by unraveling them. Phosphorylated vim NT was considerably less active in this respect. Further digestion of the rod domain of vimentin with chymotrypsin yielded 17.4 and 21 kDa polypeptides, which were tentatively characterized as originating from the carboxy- and amino-terminal half of the rod domain, respectively. Both formed salt-stable complexes with vim NT, the smaller polypeptide with a higher efficiency than the larger one. These results suggest that the staggered, antiparallel arrangement of the two coiled-coils in the protofilaments of IF proteins is, at least in part, determined by the twofold, symmetrical association of the amino-terminal head regions of one coiled-coil rope structure with the carboxy-terminal halves of the alpha-helical rod domains of the other coiled-coil and that similar interactions occur during filament assembly and in the intact filament.
Amino Acid Sequence↗
Aggregation of wool keratin intermediate filament proteins.
The wool keratin intermediate filament proteins were isolated as their S-carboxymethyl derivatives (S-carboxymethylkerateine A, SCMKA) and purified by gel filtration to remove residual non-helical protein of low molecular weight. The alpha-helix content of purified SCMKA was approximately 62% in agreement with that predicted for the alpha-helical coiled-coil segments from the amino acid sequences of the subunits. In aqueous buffer at pH 11 or in n-propanol (20% v/v) at pH 9.2 very large aggregates are dissociated and SCMKA exists largely as a mixture of the dimer (two-chain coiled-coil of Mr approximately 103,000) and the tetramer. The protein species are not in rapidly reversible equilibrium as judged from gel filtration and sedimentation equilibrium. It is probable that species with a range of association constants are present. The equilibrium is shifted towards the dimer with change of pH from 9.2 to 11 or by the addition of 20% (v/v) n-propanol. The tetrameric proteolytic digestion product which is derived from the 1B segment of the alpha-helical rod section of the keratin molecule dissociates in a similar way to intact SCMKA with increase of pH and in the presence of n-propanol. This indicates the importance of this region of the rod domain in the initial stages of the assembly of the filament. Electrostatic and hydrophobic interactions are implicated in the association of the two-chain coiled-coil to the tetramer both in intact SCMKA and the 1B segment tetramer. The results are discussed in relation to the intact dimeric and tetrameric complexes obtained from other intermediate filament types.
Amino Acids↗
Expression of intermediate filament proteins in fetal and adult human kidney: modulations of intermediate filament patterns during development and in damaged tissue.
The expression of intermediate filament proteins, particularly individual cytokeratins (CKs), vimentin, and glial filament protein, was immunohistochemically investigated using frozen sections and Carnoy-fixed, paraffin-embedded tissue from normal fetal and adult human kidneys as well as from pathologically altered kidneys. In fetal kidneys, the co-expression of CKs and vimentin was detected in the visceral and parietal epithelium of the glomerulus, the proximal tubules, the thin loops of Henle, and the collecting ducts. In contrast, in the tubules of normal adult kidneys, the presence of vimentin and CKs was nearly always mutually exclusive. While CKs 8 and 18 were present in all tubular epithelia, CKs 19 and 7 each exhibited a distinctive distribution pattern, there being a striking alteration between positive and negative segments and, not infrequently, intratubular heterogeneities. In certain segments, particular cell types (e.g., "plica cells," intercalated cells) could thus be recognized. In tubular epithelia altered by various injurious conditions, novel or enhanced expression of vimentin, CK 19 and CK 7, and, less frequently, CK 17 and glial filament protein was noted in certain segments. The increase in intermediate filament protein expression in altered (particularly proximal) tubules appeared to parallel the reduction in the degree of differentiation. Vimentin was never detected in distal tubules. The present results reveal a considerable similarity between the intermediate filament patterns in non-neoplastic proximal tubules of fetal and damaged kidney tissue and those in clear-cell and chromophilic renal cell carcinomas. They also serve to illustrate that the analysis of both fetal development and reactive cell changes may significantly contribute to our understanding of differentiation phenomena in malignant tumors.
Antibodies, Monoclonal↗
The 53kDa polypeptide component of the bovine fibre cell cytoskeleton is derived from the 115kDa beaded filament protein: evidence for a fibre cell specific intermediate filament protein.
The 115kDa protein found enriched in the PMCC (plasma membrane-cytoskeleton complex) fraction of the cortex in bovine lens fibre cells is proteolytically processed to a stable 53kDa product. The 115 kDa protein and the 53kDa polypeptide have been purified by a combination of ion exchange and hydroxyapatite chromatography. Tryptic peptide mapping using reverse phase HPLC and subsequent peptide sequencing confirmed that the 53kDa polypeptide is derived from the 115kDa protein. The 53kDa fragment is also a component of the PMCC as well as being a major component of the urea soluble fraction of lens plasma membranes which have been extracted with buffers containing 1M KC1. The 53kDa polypeptide has escaped identification as a breakdown product of the 115kDa protein because it is not recognised by a commonly used monoclonal antibody, R2D2, specific for the bovine 115kDa protein. This result suggests that proteolysis is important in determining the function(s) of the 115kDa protein, and that part of this function is satisfied by the 53kDa protein core. Both the purified 115kDa protein and the 53kDa polypeptide were unable to form either beaded or intermediate filaments on their own but they were able to form short 10nm rods indicative of an intermediate stage in intermediate filament assembly. Comparison ot the assembly properties of the 53 and 115kDa proteins indicate that there are sequences in the 115kDa protein which inhibit in vitro assembly. This is similar to the situation with neurofilament proteins. We suggest that the 115kDa protein is a lens-specific intermediate filament protein.
Amino Acid Sequence↗
Malnutrition induces an increase in intermediate filament protein content of rat cerebral cortex.
Wistar rats were fed a normal protein (25% casein) or an isoenergetic low protein (8% casein) diet from the day of giving birth until pups were weaned. Some litters were killed at weaning; others (both normal and malnourished animals) received the 25% protein diet until d 90 when they were killed. Intermediate filament (IF) preparations were obtained by extraction of the cerebral cortex with a high salt PBS solution containing 1% Triton X-100. The pellet contained the bulk of the cytoskeleton proteins from tissue, identified as the 150- and 68-kDa subunits of neurofilaments (NF-M and NF-L, respectively), the 66-kDa associated protein, the 57-kDa intermediate filament-like protein, and the 50-kDa glial fibrillary acidic protein. Intermediate filament-enriched fractions from control and malnourished rats at both d 21 and 90 were scanned following two-dimensional gel electrophoresis to determine the effects of postnatal malnutrition on the intermediate filament protein content. The results indicated that postnatal malnutrition imposed during the brain growth spurt period did not alter the expression of IF proteins of the cerebral cortex in 21-d-old rats, but increased the expression of NF-L and NF-M proteins in adult rats.
Animals↗
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↗
Aspects of the evolution of the lamin/intermediate filament protein family: a current analysis of invertebrate intermediate filament proteins.
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Animals↗
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↗
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↗
Malignant fibrous histiocytoma. Heterogeneous patterns of intermediate filament proteins by immunohistochemistry.
Patterns of intermediate filament expression of 10 malignant fibrous histiocytomas (MFHs) were immunohistochemically evaluated using acetone-fixed frozen sections. Seven cases represented the storiform-pleomorphic subtype, 2 were of myxoid type, and 1 was of giant-cell type. All cases had been studied by electron microscopy, and no proof for the diagnoses of liposarcoma, rhabdomyosarcoma, and leiomyosarcoma could be obtained. All tumors showed prominent vimentin immunoreactivity in the tumor cells. Cytokeratin-positive neoplastic cells were found in 2 cases, and in the majority of tumor cells in 1 of these. The 68k neurofilament-positive cells were found in 2 cases. Desmin was not found beyond doubt in the neoplastic cells in any cases, and all cases were negative for glial fibrillary acidic protein. The expression of several types of intermediate filament indicates divergent differentiation properties in MFH and may suggest the heterogeneity of this entity, but more cases should be studied to elaborate any possible consistent patterns of intermediate filament expression in different types of MFH. The expression of multiple types of intermediate filament proteins in MFH can alternatively signify random activation of the corresponding genes in the primitive tumor cells. The complex patterns of intermediate filament proteins in morphologically defined MFHs should be taken into account in the practical immunohistologic analysis of tumors.
Cell Differentiation↗
Exoenzyme S of Pseudomonas aeruginosa ADP-ribosylates the intermediate filament protein vimentin.
Exoenzyme S, which had been thought to be unselective, catalyzes the ADP-ribosylation of only a subset of cellular proteins. The intermediate filament protein vimentin is one of the more abundant substrates. Disassembled vimentin, and proteolytic fragments of vimentin that cannot form filaments, is more readily ADP-ribosylated than is filamentous vimentin.
ADP Ribose Transferases↗
Structure of the mouse gene encoding peripherin: a neuronal intermediate filament protein.
The gene encoding mouse peripherin, a neuronal intermediate filament protein, has been cloned. Its sequence, through 1021 nucleotides composing the 5'-flanking region, nine exons, eight introns and 547 nucleotides of the 3'-flanking region, as well as its transcription initiation site have been determined. The amino acid coding sequence differs from that of the rat peripherin gene. The mouse gene has an additional histidine near the N-terminal end, and shows three conservative and two non-conservative changes. The promoter sequence, containing the binding sites for transcription factors as well as other sequences is homologous to promoter regions of other type III intermediate filament protein genes and other neuronal-specific genes.
Amino Acid Sequence↗
Identification and developmental expression of a novel low molecular weight neuronal intermediate filament protein expressed in Xenopus laevis.
Xenopus laevis is a valuable model system for the study of vertebrate neuroembryogenesis. However, very few well-characterized nervous system-specific molecular markers are available for studies in this organism. We screened a X. laevis adult brain cDNA library using a cDNA probe for mouse low molecular weight neurofilament protein (NF-L) in order to identify neuron-specific intermediate filament proteins. Clones for two distinct neuron-specific intermediate filament proteins were isolated and sequenced. One of these encoded for a Xenopus NF-L (XNF-L) and the other for a novel neuron-specific Xenopus intermediate filament protein (XNIF) that was present earlier and more abundantly than XNF-L during development. XNIF contained a central rod domain with multiple sequence features characteristic of IF proteins. The XNF-L was very similar to mouse NF-L, with a 77% sequence identity in the rod domain and the presence of a polyglutamic acid region in the tail domain, characteristic of type IV neurofilament proteins. In contrast, XNIF showed only 60% identity to mouse NF-L in the rod domain and lacked the glutamic acid-rich sequence in the tail domain. XNIF also had a very low (approximately 38%) sequence identity in the head and tail domains as compared to NF-L and other neurofilament proteins (45% identity to the head domain of alpha-internexin). In the adult frog, XNIF mRNA is detected by Northern blots only within the nervous system and by in situ hybridization histochemistry exclusively in neurons, particularly in the medullary reticular system and spinal cord. Antisera raised against the unique tail region of XNIF detected a single distinct 60 kDa band in Western blots of nervous system cytoskeletal preparations, and this XNIF immunoreactivity was concentrated in axons in the PNS and in small perikarya in the dorsal root ganglion. In contrast, NF-L immunoreactivity was principally in the large perikarya in the dorsal root ganglion. In development, XNIF mRNA appears more abundant than XNF-L mRNA in all premetamorphic stages examined. XNIF mRNA is first detectable at stage 24 (26 hr), whereas stable expression of XNF-L is at stage 35/36 (50 hr). XNIF immunoreactivity is detectable within the cement gland, within many neuronal cell bodies and axon tracts within the developing nervous system, and within all cellular layers of the developing retina. The availability of these two distinct neuron-specific intermediate filament proteins, with different temporal and spatial expression patterns, should provide new markers as well as targets for functional perturbation in the developing X. laevis nervous system.
Amino Acid Sequence↗