Search

Objective: To screen and identify the genes related to the occurrence and development of varicose great saphenous vein in the patients with primary deep vein valve insufficiency (PDVI)., Methods: mRNA fluorescent differential display (FDD) technique was used to compare the different cDNA fragments originated from differentially expressed mRNAs from the venous tissues of 10 patients with varicose great saphenous vein complicated with PDVI. Ten specimens of normal venous tissue from 10 patients dying from other diseases were used as controls. The differently expressed cDNA fragments were then re-amplified and labeled with DIG to prepare probes for later Northern blotting. Positive fragments confirmed by Northern blotting were cloned into pGEM-T easy vector and sequenced using Sanger's method. Then the sequences were compared with the data in GeneBank by BLASTN software to search for their genetic origin., Results: Altogether 37 differentially expressed cDNA fragments were discovered from the 2 groups, among which 30 were confirmed by Northern blotting. There was a notable 540 bp-long cDNA fragment, which was only presented in the control group, sharing 99% homology with part of the mRNA sequence of human KIAA0353 gene., Conclusion: The varicose great saphenous vein of PDVI patients is a process with the involvement of multiple genes and the default of KIAA0353 gene may play a role in the occurrence and development of varicose great saphenous vein in PDVI patients.

The isolation from proliferating mouse and human embryo fibroblasts of SDS-stable crosslinkage products of vimentin with DNA fragments containing inverted repeats capable of cruciform formation under superhelical stress and the competitive effect of a synthetic Holliday junction on the binding of cytoplasmic intermediate filament (cIF) proteins to supercoiled DNA prompted a detailed investigation of the proteins' capacity to associate with four-way junction DNA and to influence its processing by junction-resolving endonucleases. Electrophoretic mobility shift analysis of reaction products obtained from vimentin and Holliday junctions under varying ionic conditions revealed efficient complex formation of the filament protein not only with the unstacked, square-planar configuration of the junctions but also with their coaxially stacked X-conformation. Glial fibrillary acidic protein (GFAP) was less efficient and desmin virtually inactive in complex formation. Electron microscopy showed binding of vimentin tetramers or octamers almost exclusively to the branchpoint of the Holliday junctions under physiological ionic conditions. Even at several hundredfold molar excess, sequence-related single- and double-stranded DNAs were unable to chase Holliday junctions from their complexes with vimentin. Vimentin also stimulated bacteriophage T7 endonuclease I in introducing single-strand cuts diametrically across the branchpoint and thus in the resolution of the Holliday junctions. This effect is very likely due to vimentin-induced structural distortion of the branchpoint, as suggested by the results of hydroxyl radical footprinting of Holliday junctions in the absence and the presence of vimentin. Moreover, vimentin, and to a lesser extent GFAP and desmin, interacted with the cruciform structures of inverted repeats inserted into a supercoiled vector plasmid, thereby changing their configuration via branch migration and sensibilizing them to processing by T7 endonuclease I. This refers to both plasmid relaxation caused by unilateral scission and, particularly, linearization via bilateral scission at primary and cIF protein-induced secondary cruciform branchpoints that were identified by T7 endonuclease I footprinting. cIF proteins share these activities with a variety of other architectural proteins interacting with and structurally modulating four-way DNA junctions. In view of the known and hypothetical functions of four-way DNA junctions and associated protein factors in DNA metabolism, cIF proteins as complementary nuclear matrix proteins may play important roles in such nuclear matrix-associated processes as DNA replication, recombination, repair, and transcription, with special emphasis on both the preservation and evolution of the genome.

Outer dense fibres (ODF) are important substructures of mammalian sperm tails that are involved in the regulation of sperm motility. In this study, we investigated the identity of several sodium dodecyl sulphate (SDS)-insoluble ODF proteins. Bovine ODF were purified by separating sperm heads and tails using ultrasound and Percoll(R) density gradient centrifugation. Sperm flagella were treated with the detergent cetyltrimethylammonium bromide (CTAB). CTAB-insoluble material, which reportedly represents the ODF fraction, was collected, and electron microscopy confirmed a highly purified ODF fraction. We found after solubilization of this fraction with SDS that high amounts of insoluble material were retained after centrifugation. SDS-insoluble material was collected and quantitatively dissolved in 8 M urea. SDS-gel electrophoresis in the presence of urea revealed polypeptides with apparent molecular masses of approximately 25, 43, and 50 kDa. Subsequent immunoblotting with anti-cytokeratin antibodies detected two urea-soluble, SDS-insoluble proteins with apparent molecular masses of 45 and 66 kDa. The 45-kDa protein was identified as cytokeratin 19. An antibody reacting with a palette of cytokeratins (CK 1-18 and CK 20), KL1, was the only antibody that reacted with the 66-kDa polypeptide. We conclude that sperm ODF fractions contain at least one each of type I and type II intermediate filaments. As keratins and intermediate filaments are described as rope-like structures, we suggest that these intermediate filaments play an important structural or tension-bearing role in sperm flagella.

The selection of DNA fragments containing simple d(GT)(n) and composite d(GT)(m). d(GA)(n) microsatellites during affinity binding of mouse genomic DNA to type III cytoplasmic intermediate filaments (cIFs) in vitro, and the detection of such repeats, often as parts of nuclear matrix attachment region (MAR)-like DNA, in SDS-stable DNA-vimentin crosslinkage products isolated from intact fibroblasts, prompted a detailed study of the interaction of type III cIF proteins with left-handed Z-DNA formed from d(GT)(17) and d(CG)(17) repeats under the topological tension of negatively supercoiled plasmids. Although d(GT)(n) tracts possess a distinctly lower Z-DNA-forming potential than d(CG)(n) tracts, the filament proteins produced a stronger electrophoretic mobility shift with a plasmid carrying a d(GT)(17) insert than with plasmids containing different d(CG)(n) inserts, consistent with the facts that the B-Z transition of d(GT)(n) repeats requires a higher negative superhelical density than that of d(CG)(n) repeats and the affinity of cIF proteins for plasmid DNA increases with its superhelical tension. That both types of dinucleotide repeat had indeed undergone B-Z transition was confirmed by S1 nuclease and chemical footprinting analysis of the plasmids, which also demonstrated efficient protection by cIF proteins from nucleolytic and chemical attack of the Z-DNA helices as such, as well as of the flanking B-Z junctions. The analysis also revealed sensibilization of nucleotides in the center of one of the two strands of a perfect d(CG)(17) insert toward S1 nuclease, indicating cIF protein-induced bending of the repeat. In all these assays, vimentin and glial fibrillary acidic protein (GFAP) showed comparable activities, versus desmin, which was almost inactive. In addition, vimentin and GFAP exhibited much higher affinities for the Z-DNA conformation of brominated, linear d(CG)(25) repeats than for the B-DNA configuration of the unmodified oligonucleotides. While double-stranded DNA was incapable of chasing the Z-DNA from its protein complexes, and Holliday junction and single-stranded (ss)DNA were distinguished by reasonable competitiveness, phosphatidylinositol (PI) and, particularly, phosphatidylinositol 4,5-diphosphate (PIP(2)) turned out to be extremely potent competitors. Because PIP(2) is an important member of the nuclear PI signal transduction cascade, it might exert a regulatory influence on the binding of cIF proteins to Z- and other DNA conformations. From this interaction of cIF proteins with Z- and bent DNA and their previously detected affinities for MAR-like, ss, triple helical, and four-way junction DNA, it may be concluded that the filament proteins play a general role in such nuclear matrix-associated processes as DNA replication, recombination, repair, and transcription.

The various plectin isoforms are among the major crosslinking elements of the cytoskeleton. The importance of plectin in epithelia is convincingly supported by the severe skin blistering observed in plectin-deficient humans and mice. Here, we identified plectin 1a (> 500 kDa), a full length plectin variant containing the sequence encoded by the alternative first exon 1a, as the isoform most prominently expressed in human and mouse keratinocytes. In skin sections and cultured keratinocytes, plectin 1a was shown to colocalize with hemidesmosomal structures. In contrast, a second isoform expressed in epithelia, plectin 1c, differing from 1a merely by a short N-terminal sequence, colocalized with microtubules. Expression of plectin 1a, but not of its N-terminal fragment alone, or of a third alternative full length isoform (plectin 1), restored the reduced number of hemidesmosome-like stable anchoring contacts in cultured plectin-null keratinocytes. Our results show for the first time that different isoforms of a cytolinker protein expressed in one cell type perform distinct functions. Moreover, the identification of plectin 1a as the isoform defects in which cause skin blistering in plectin-related genetic diseases, such as epidermolysis bullosa simplex MD and epidermolysis bullosa simplex Ogna, could have implications for the future development of clinical therapies for patients.

Filaggrin is a keratin filament associated protein that is expressed in granular layer keratinocytes and derived by sequential proteolysis from a polyprotein precursor termed profilaggrin. Depending on the species, each profilaggrin molecule contains between 10 and 20 filaggrin subunits organized as tandem repeats with a calcium-binding domain at the N- terminal end. We now report the characterization of the complete mouse gene. The structural organization of the mouse gene is identical to the human profilaggrin gene and consists of three exons with a 4 kb intron within the 5' noncoding region and a 1.7 kb intron separating the sequences encoding the calcium-binding EF-hand motifs. A processed pseudogene was found embedded within the second intron. The third and largest exon encodes the second EF-hand, a basic domain (designated the B-domain) followed by 12 filaggrin repeats and a unique C-terminal tail domain. A polyclonal antibody raised against the conceptually translated sequence of the B-domain specifically stained keratohyalin granules and colocalized with a filaggrin antibody in granular layer cells. In upper granular layer cells, B-domain containing keratohyalin granules were in close apposition to the nucleus and, in some cells, appeared to be completely engulfed by the nucleus. In transition layer cells, B-domain staining was evident in the nucleus whereas filaggrin staining remained cytoplasmic. Nuclear staining of the B-domain was also observed in primary mouse keratinocytes induced to differentiate. This study has also revealed significant sequence homology between the mouse and human promoter sequences and in the calcium-binding domain but the remainder of the protein-coding region shows substantial divergence.

To identify phosphoproteins that might play a role in naringin-sensitive hepatocellular cytoskeletal disruption and apoptosis induced by algal toxins, hepatocyte extracts were separated by gel electrophoresis and immunostained with a phosphothreonine-directed antibody. Use of dilute (5%) polyacrylamide gels containing 6 m urea allowed the resolution of one very large (approximately 500-kDa) okadaic acid- and naringin-sensitive phosphoprotein, identified by tryptic fingerprinting, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and immunostaining as the cytolinker protein, plectin. The naringin-sensitive phosphorylation induced by okadaic acid and microcystin-LR probably reflected inhibition of a type 2A protein phosphatase, whereas the naringin-resistant phosphorylation induced by calyculin A, tautomycin, and cantharidin probably involved a type 1 phosphatase. Okadaic acid caused a collapse of the plectin-immunostaining bile canalicular sheaths and the general cytoskeletal plectin network into numerous medium-sized plectin aggregates. Inhibitors of protein kinase C, cAMP-dependent protein kinase, or Ca(2+)/calmodulin-dependent kinase II had moderate or no protective effects on plectin network disruption, whereas naringin offered 86% protection. Okadaic acid induced a naringin-sensitive phosphorylation of AMP-activated protein kinase (AMPK), the stress-activated protein kinases SEK1 and JNK, and S6 kinase. The AMPK-activating kinase (AMPKK) is likely to be the target of inhibition by naringin, the other kinases serving as downstream components of an AMPKK-initiated signaling pathway.

Profilaggrin is expressed in the differentiating granular layer of epidermis and other stratified epithelia, where it forms a major component of cytoplasmic keratohyalin granules. It consists of two distinct domains, an N-terminal S100-like Ca2+- binding domain containing two EF-hands and multiple filaggrin units that aggregate keratin filaments in the stratum corneum. Here, we report structure-function studies of the N-terminal peptide from mouse, human, and rat profilaggrin. The profilaggrin N- terminal peptides of all species contain two S100-like EF-hands, bipartite nuclear localization sequences, and proprotein convertase cleavage sites. The nuclear localization signals in human and mouse profilaggrin were shown to be functional by transfection of epithelial cells and depended on the absence of filaggrin sequences. The nuclear localization of the processed (free) N-terminal peptide of human profilaggrin is consistent with immunolocalization findings in normal human skin and in parakeratotic skin disorders, which exhibit nuclear staining of granular and/or cornified layers. The mouse profilaggrin N-terminus undergoes proteolytic processing in two steps, first releasing an N-terminal peptide containing some filaggrin sequence and finally the free N-terminus of 28-30 kDa; these peptides have cytoplasmic and nuclear distributions, respectively, when expressed in transfected cells. The N-terminal processing may occur prior to or simultaneously with the proteolytic processing of the polyfilaggrin domain. The nuclear accumulation of the profilaggrin N-terminal peptide in epidermis and in transfected cells strongly suggests a calcium-dependent nuclear function for the profilaggrin N-terminus during epidermal terminal differentia tion when the free N-terminus is released from profilaggrin by specific proteolysis.

Intermediate filament (IF) proteins form the largest family of cytoskeletal proteins in mammalian cells. The function of these proteins has long been thought to be only structural. However, this single function does not explain their diverse tissue- and differentiation-specific expression patterns. Evidence is now emerging that IF also act as an important framework for the modulation and control of essential cell processes, in particular, signal transduction events. Here, we review the most recent developments in this growing and exciting new field., (Copyright 2002 Wiley Periodicals, Inc.)

Plectin is a cyoskeletal linker protein that protects tissues against mechanical stress. We report here that the N-terminal domain of the nonreceptor tyrosine kinase Fer interacts with N-terminal sequences of plectin. Recombinant protein encoded by exon 12-24 of rat plectin bound directly to amino acid 1-329 of murine Fer. Using an antiserum prepared to a recombinant N-terminal fragment of Fer kinase, plectin was coimmunoprecipitated with Fer from cell lysates of cultured mouse fibroblasts. Plectin was shown to partially colocalize with Fer in these cells. Upon transfection of full length Fer cDNA into plectin-negative mouse fibroblasts, hyperphosphorylation of Fer was observed; hyperphosphorylation was strongly reduced when N-terminal Fer deletion mutants were transfected. Immunocomplex kinase assays showed that the activity of Fer kinase transfected into plectin-negative fibroblasts was increased compared to that transfected into wild type cells. We conclude that Fer interacts with plectin and that this interaction may serve to negatively regulate Fer's activity.

Plectin is an abundantly expressed cytoskeletal crosslinking protein of enormous size (>500 kDa) and multiple functions. It represents one of the many members of a large family of actin-binding proteins. The actin-binding domain of mouse plectin was expressed in Escherichia coli and purified to homogeneity. Crystals of the actin-binding domain of plectin were prepared by the hanging-drop method. They belong to space group P2(1), with unit-cell parameters a = 55.92, b = 108.92, c = 63.75 A, beta = 115.25 degrees. Data from a single crystal were collected to 2.0 A resolution at room temperature using synchrotron radiation at EMBL, Hamburg. The asymmetric unit contains two molecules of the protein, which corresponds to V(M) = 3.06 A(3) Da(-1) and a solvent content of 60%. The structure was solved by the molecular-replacement method. In addition, the preparation of selenomethionine-derivative crystals is described.

Objective: To identify immunodominant epitopes in natural filaggrin that are reactive with antifilaggrin autoantibodies (AFA) in the sera of patients with rheumatoid arthritis (RA) and to explore their use in a diagnostic assay format., Methods: Based on the results of epitope mapping of human natural filaggrin as well as molecular modeling and computational chemistry, synthetic peptides together with recombinant citrullinated filaggrin were evaluated by a line immunoassay (LIA) for AFA detection. Diagnostic performance was assessed using 336 RA and 253 disease control sera and was compared with that of reference methods., Results: Several immunoreactive epitopes were identified in natural filaggrin, all of which contained at least 1 citrulline residue. Three antigenic substrates, including 2 synthetic peptides and recombinant citrullinated filaggrin showing maximal reactivity on LIA, were finally selected. Using the 3-antigen LIA3, overall sensitivity, specificity, and positive predictive value for RA were 65.2%, 98.0%, and 89.1%, respectively, compared with 61.9%, 98.8%, and 92.8% using the 2-antigen LIA2 (without recombinant protein). Thirty-seven percent of the rheumatoid factor (RF)-negative RA samples (30 of 81) were AFA-positive by LIA2, and 52 of 54 RF-positive control samples had no AFA detected on LIA2. Higher specificity and sensitivity were obtained by LIA2 versus anti-RA33 immunoblot, whereas good agreement was observed with antikeratin antibody testing. LIA performed significantly better than AFA immunoblotting using natural filaggrin, at a specificity level of 99% (P = 0.0047)., Conclusion: Citrullinated residues are present in immunoreactive epitopes of natural human filaggrin. AFA can be readily detected by citrullinated peptides in an LIA-based test, resulting in high specificity and positive predictive value for RA. The LIA could serve as a user-friendly alternative to existing immunofluorescence tests and AFA immunoblot techniques. Given its complementarity to RF, this test can be a valuable tool in the differential diagnosis of arthritis.

The urochordate Ciona intestinalis is a well established system for embryological studies, and large scale EST sequences begin to emerge. We cloned five cytoplasmic intennediate filament (IF) cDNAs and made specific antibodies to the recombinant proteins. Self-assembly studies and immunofluorescence microscopy were used to study these proteins and their distribution. Confirming and extending previous studies in Styela, we found that Ciona protein IF-A is expressed in muscle and forms homopolymeric filaments while proteins IF-C and IF-D, which form only obligatory heteropolymeric filaments, resemble a keratin pair exclusively found in the entire epidermis. Protein IF-B and the new protein IF-F potentially reflect tunicate-specific IF proteins. They are found in the entire internal epithelia including the neural gland. We also extended the analysis to earlier developmental stages of Ciona. Protein IF-A is expressed in muscle from larval stages, whereas proteins IF-C and IF-D are found only in the tail epidermis. Protein IF-F is detected abundantly in the test cells of eggs, embryos and premetamorphic larvae. Our studies show that IF proteins could prove very useful markers in the study of cell fate determination in Ciona. They also support previous findings on the evolutionary relationships of different IF proteins. Non-vertebrate chordates have IF proteins which represent orthologs of vertebrate type I to III proteins, but also IF proteins that do not seem to fit into these classes. However, the intron positions of all tunicate IF genes are conserved with vertebrate type I to III genes, pointing to a common evolutionary origin.

The two small intermediate filament (IF) proteins A3 and B2 of the cephalochordate Amphioxus were investigated. Blot overlays indicated a heterotypic interaction pattern of the recombinant proteins. While the individual proteins formed only aggregates, the stoichiometric mixture formed obligatory heteropolymeric filaments. Mutant proteins with a single cysteine residue in equivalent positions gave rise to filaments that oxidize to the disulfide-linked heterodimer, which can again form IF. Thus the A3/B2 filaments, which are expressed in the intestinal epithelium, are based on a hetero coiled coil. This keratin-like assembly process of A3 plus B2 was unexpected, since previous evolutionary tree calculations performed by two laboratories on the various Amphioxus IF proteins identified keratin I and II orthologs but left the A/B group as a separate branch. We discuss obvious evolutionary aspects of the Amphioxus IF multigene family, including the previously made observation that B1, the closest relative of B2, forms homopolymeric IF in vitro and is, like vertebrate type III proteins, expressed in mesodermally derived tissues., (Copyright 2002 Elsevier Science Ltd.)

We recently identified a novel protein called syncoilin, a putative intermediate filament protein that interacts with alpha-dystrobrevin, a member of the dystrophin-associated protein complex. Syncoilin is found at the neuromuscular junction, sarcolemma, and Z-lines and is thought to be important for muscle fiber integrity. Based on the similar protein structure and cellular localization of syncoilin and desmin, we proposed that these proteins interact in vivo. The data presented confirm an interaction between syncoilin and desmin and demonstrate their co-localization in skeletal muscle. Intriguingly, whereas these proteins interact, COS-7 cell expression studies show that desmin and syncoilin do not assemble into heterofilaments. Furthermore, fractionation assay and immunofluorescence study of H2K myoblasts and myotubes suggest that, unlike typical intermediate filament proteins, syncoilin does not participate in filament formation with any protein. However, it is possible that syncoilin is involved in the anchoring of the desmin intermediate filament network at the sarcolemma and the neuromuscular junction. This interaction is likely to be important for maintaining muscle fiber integrity and may also link the dystrophin-associated protein complex to the cytoskeleton. The dysfunction or absence of syncoilin may result in the disruption of the intermediate filament network leading to muscle necrosis. Syncoilin is therefore an ideal candidate gene for muscular dystrophies and desmin-related myopathies.

By connecting cytoskeletal elements to each other and to junctional complexes, the plakin family of cytolinkers plays a crucial role in orchestrating cellular development and maintaining tissue integrity. Plakins are built from combinations of interacting domains that bind to microfilaments, microtubules, intermediate filaments, cell-adhesion molecules and members of the armadillo family. Plakins are involved in both inherited and autoimmune diseases that affect the skin, neuronal tissue, and cardiac and skeletal muscle. Here, we describe the members of the plakin family and their interaction partners, and give examples of the cellular defects that result from their dysfunction.

A novel mouse cDNA named hornerin was isolated by RNA differential display applied to developing mouse skin. Hornerin, which has 2,496 amino acids, comprises EF-hand domains at the N terminus followed by a spacer sequence and a large repetitive domain, indicating that hornerin is a novel member of the "fused gene"-type cornified envelope precursor protein family. The repetitive domain of hornerin was found to be rich in glycine, serine, and glutamine. Hornerin was expressed in the tongue, esophagus, forestomach, and skin among the adult mouse tissues examined, all of them cornifying stratified epithelium. In the embryonic mouse skin, hornerin mRNA was first detected on gestational day 15.5 in the epidermis coincidentally with the formation of a granular layer. In accordance with this, hornerin was detected in the granular and cornified layers of the mature epidermis. In the granular cells of the epidermis, the hornerin protein was detected in keratohyalin granules together with profilaggrin. Furthermore, Western blot analysis of the mouse skin showed that the hornerin protein was cleaved during the process of epidermal differentiation, indicating possible posttranslational proteolytic processing as is observed in profilaggrin. Differentiation of primary mouse epidermal keratinocytes with 0.12 mm Ca(2+) resulted in the induction of hornerin. These results indicate that hornerin is structurally as well as functionally most similar to profilaggrin among the family members and possibly plays pleiotropic roles, including a role in cornification.

Intermediate filament (IF) proteins are constituents of the cytoskeleton, conferring resistance to mechanical stress, and are encoded by a dispersed multigene family. In man we have identified two isoforms (180 and 150 kDa) of the IF protein synemin. Synemin alpha and beta have a very short N-terminal domain of 10 amino acids and a long C-terminal domain consisting of 1243 amino acids for the alpha isoform and 931 amino acids for the beta isoform. An intronic sequence of the synemin beta isoform is used as a coding sequence for synemin alpha. Both mRNA isoforms (6.5 and 7.5 kb) result from alternative splicing of the same gene, which has been assigned to human chromosome 15q26.3. Analyses by Northern and Western blot revealed that isoform beta is the predominant isoform in striated muscles, whereas both isoforms (alpha and beta) are present in almost equal quantities in smooth muscles. Co-transfection and immunolabeling experiments indicate that both synemin isoforms are incorporated with desmin to form heteropolymeric IFs. Furthermore synemin and desmin are found aggregated together in certain pathological situations.

Peripherin/rds is an integral membrane protein required for the elaboration of rod and cone photoreceptor outer segments in the vertebrate retina; it causes a surprising variety of progressive retinal degenerations in humans and dysmorphic photoreceptors in murine models if defective or absent. (Peripherin/rds is also known as photoreceptor peripherin, peripherin/rds, rds/peripherin, rds, and peripherin-2.) Peripherin/rds appears to act as a structural element in outer segment architecture. However, neither its function at the molecular level nor its role in retinal disease processes are well understood. This report initiates a systematic investigation of protein domain structure and function by examining the molecular and cellular consequences of a series of 14 insertional mutations distributed throughout the polypeptide sequence. Protein expression, disulfide bonding, sedimentation velocity, and subcellular localization of the COS-1 cell-expressed mutant variants were examined to test the hypothesis that protein folding and tetrameric subunit assembly are mediated primarily by EC2, a conserved extracellular/intradiskal domain. Protein folding and tetrameric subunit assembly were not affected by insertion of either an uncharged dipeptide (GA) or a highly charged hendecapeptide (GDYKDDDDKAA) into any one of nine sites residing outside of EC2 as assayed by nonreducing Western blot analysis, sedimentation velocity, and subcellular localization. In contrast, insertions at five positions within the EC2 domain did cause either gross protein misfolding (two sites) or a reduction in protein sedimentation coefficient (two sites) or both (one site). These results indicate that although the vast majority of extramembranous polypeptide sequence makes no measurable contribution to protein folding and tetramerization, discrete regions within the EC2 domain do contain determinants for normal subunit assembly. These findings raise the possibility that multiple classes of structural perturbation are produced by inherited defects in peripherin/rds and contribute to the observed heterogeneity of retinal disease phenotypes.

Plectin, a member of the cytolinkers protein family, plays a crucial role in cells as a stabilizing element of cells against mechanical stress. Its absence results in muscular dystrophy, skin blistering, and signs of neuropathy. The C-terminal domain of plectin contains several highly homologous repeat domains that also occur in other cytolinkers. Secondary structure analysis revealed that the building block of these domains, the PLEC repeat, is similar to the ankyrin repeat. We present a model that attempts to explain how the C-terminal domain, which comprises approximately 1900 amino acid, could be stabilized to maintain its structural integrity even under extensive mechanical stress. In this model, larger solenoid modules formed from PLEC repeats can be disulfide-bridged via conserved cysteines. Our hypothesis suggests that this process could be mediated by cytoplasmic NOS-generated products, such as the radical peroxynitrite. Reinforcement of molecular structure would provide a rationale why during exercising or physical stress radicals are formed without necessarily being deleterious. This article contains supplementary material that may be viewed at the BioEssays website at http://www.interscience.wiley.com/jpages/0265-9247/suppmat/23/v23_11.1064.html., (Copyright 2001 John Wiley & Sons, Inc.)

Complementary DNA (cDNA) corresponding to mouse nestin intermediate filament protein, a specific marker for neural stem cells, was isolated and characterized. The complete sequence comprised 5983 base pairs encoding 1821 amino acids, and the deduced polypeptide was similar to rat (84%), hamster (73%), and human (62%) nestin. Southern blots showed that mouse nestin was a single-copy gene, and Northern blots detected a 6.0 kilobase mRNA transcript. When the cDNA was overexpressed as an enhanced green fluorescent fusion protein in COS7 cells, nestin immunoreactivity appeared in the filamentous cytoskeletal network. Accordingly, biologically active mouse nestin cDNA may offer an important new tool for stem cell research.

A combination of enzymatic and chemical ladder sequencing of photo-cross-linked protein-single-stranded oligodeoxyribonucleotide complexes and analysis by MALDI-TOF mass spectrometry was employed to identify the amino acid residues responsible for the stable binding of nucleic acids in several intermediate filament (IF) subunit proteins. The IF proteins studied included the type I and type II cytokeratins K8, K18, and K19; the type III proteins desmin, glial fibrillary acidic protein (GFAP), peripherin, and vimentin; and the type IV neurofilament triplet protein L (NF-L). The site of nucleic acid binding was localized to the non-alpha-helical, amino-terminal head domain of all of the IF proteins tested. GFAP, which has the shortest head domain of the proteins tested, cross-linked via only two amino acid residues. One of these residues was located within a conserved nonapeptide domain that has been shown to be required for filament formation. One or more cross-linked residues were found in a similar location in the other proteins studied. The major binding site for nucleic acids for most of the proteins appears to be localized within the middle of the head domain. The two exceptions to this generalization are GFAP, which lacks these residues, and NF-L, in which a large number of cross-linked residues were found scattered throughout the first half of the head domain. Control experiments were also done with two bacteriophage ssDNA-binding proteins, as well as actin and tubulin. The single sites of cross-linkage observed with the bacteriophage proteins, Phe(183) for the T4 gene 32 protein and Phe(73) for the M13 gene 5 protein, were in good agreement with literature data. Actin and tubulin could not be cross-linked to the oligonucleotide. Aside from the insight into the biological activity of IF proteins that these data provide, they also demonstrate that this analytical method can be employed to study a variety of protein-nucleic acid interactions.

The spermatid nucleus and cytoplasm undergo dramatic morphological modifications during spermatid differentiation into mature sperm. Some of the external force causing this nuclear shaping is generated by a microtubular structure termed the manchette, which attaches to the perinuclear ring of the spermatid. Here, we report the isolation and characterization of a protein component of this perinuclear ring in an immunological screening of a mouse testis cDNA library. We termed this protein CLIP-50 because of its high similarity at the amino acid level to the C-terminal region of the microtubule-binding protein CLIP-170/restin. CLIP-50 lacks the characteristic microtubule-binding motif, but retains a portion of the predicted coiled-coiled domain and the metal-binding motif. The CLIP-50 transcript and protein are abundant in testis. The protein is also expressed in heart, lung, kidney, and skin. A distinct size variant exists in brain. In the spermatids, CLIP-50 protein localizes specifically to the centriolar region where the sperm tail originates and to the perinuclear ring from which the manchette emerges. CLIP-50 staining is retained in the ring throughout its migration over the surface of the nucleus which accompanies the nuclear shaping into its characteristic sperm configuration. This localization pattern indicates a very specific function for this novel CLIP derivative during mouse spermiogenesis., (Copyright 2001 Academic Press.)

The molecular interactions responsible for nuclear envelope assembly after mitosis are not well understood. In this study, we demonstrate that a peptide consisting of the COOH-terminal domain of Xenopus lamin B3 (LB3T) prevents nuclear envelope assembly in Xenopus interphase extracts. Specifically, LB3T inhibits chromatin decondensation and blocks the formation of both the nuclear lamina-pore complex and nuclear membranes. Under these conditions, some vesicles bind to the peripheral regions of the chromatin. These "nonfusogenic" vesicles lack lamin B3 (LB3) and do not bind LB3T; however, "fusogenic" vesicles containing LB3 can bind LB3T, which blocks their association with chromatin and, subsequently, nuclear membrane assembly. LB3T also binds to chromatin in the absence of interphase extract, but only in the presence of purified LB3. Additionally, we show that LB3T inhibits normal lamin polymerization in vitro. These findings suggest that lamin polymerization is required for both chromatin decondensation and the binding of nuclear membrane precursors during the early stages of normal nuclear envelope assembly.

Plectin is a major component of the cytoskeleton and is expressed in a wide variety of cell types. It plays an important role in the integrity of the cytoskeleton by cross-linking the three filamentous networks and stabilizing cell-matrix and cell-cell contacts. Sequence analysis showed that plectin contains a highly conserved actin-binding domain, consisting of a pair of calponin-like subdomains. Using yeast two-hybrid assays in combination with in vitro binding experiments, we demonstrate that the actin-binding domain of plectin is fully functional and preferentially binds to polymeric actin. The sequences required for actin binding were identified at the C-terminal end of the first calponin homology domain within the actin-binding domain of plectin. We found that the actin-binding domain of plectin is able to bundle actin filaments and we present evidence that this is mediated by the dimerization of this domain. In addition we also show that plectin and another member of the plakin family, dystonin, can heterodimerize by their actin-binding domains. We propose a new mechanism by which plectin and possibly also other actin-binding proteins can regulate the organization of the F-actin network in the cell.

The expression of distinct keratin pairs during epidermal differentiation is assumed to fulfill specific and essential cytoskeletal functions. This is supported by a great variety of genodermatoses exhibiting tissue fragility because of keratin mutations. Here, we show that the loss of K10, the most prominent epidermal protein, allowed the formation of a normal epidermis in neonatal mice without signs of fragility or wound-healing response. However, there were profound changes in the composition of suprabasal keratin filaments. K5/14 persisted suprabasally at elevated protein levels, whereas their mRNAs remained restricted to the basal keratinocytes. This indicated a novel mechanism regulating keratin turnover. Moreover, the amount of K1 was reduced. In the absence of its natural partner we observed the formation of a minor amount of novel K1/14/15 filaments as revealed by immunogold electron microscopy. We suggest that these changes maintained epidermal integrity. Furthermore, suprabasal keratinocytes contained larger keratohyalin granules similar to our previous K10T mice. A comparison of profilaggrin processing in K10T and K10(-/-) mice revealed an accumulation of filaggrin precursors in the former but not in the latter, suggesting a requirement of intact keratin filaments for the processing. The mild phenotype of K10(-/-) mice suggests that there is a considerable redundancy in the keratin gene family.

Specific proteolysis plays an important role in the terminal differentiation of keratinocytes in the epidermis and several types of proteases have been implicated in this process. The proprotein convertases (PCs) are a family of Ca2+-dependent serine proteases involved in processing and activation of several types of substrates. In this study we examined the expression and some potential substrates of PCs in epidermis. Four PCs are expressed in epidermis: furin, PACE4, PC5/6 and PC7/8. Furin is detected in two forms, either with or without the transmembrane domain, suggesting occurrence of post-translational cleavage to produce a soluble enzyme. In addition the furin active site has differential accessibility in the granular layer of the epidermis relative to the basal layer, whereas antibodies to the transmembrane domain stain both layers. These findings suggest that furin has access to different types of substrates in granular cells as opposed to basal cells. PC7/8, in contrast, is detected throughout the epidermis with antibodies to both the transmembrane and active site and no soluble form observed. A peptide PC inhibitor (dec-RVKR-CMK) inhibits cleavage of Notch-1, a receptor important in cell fate determination that is found throughout the epidermis. Profilaggrin, found in the granular layer, is specifically cleaved by furin and PACE4 in vitro at a site between the amino terminus and the first filaggrin repeat. This work suggests that the PCs play multiple roles during epidermal differentiation.

The intermediate filament protein nestin is expressed during early stages of development in the central nervous system and in muscle tissues. Nestin expression is associated with morphologically dynamic cells, such as dividing and migrating cells. However, little is known about regulation of nestin during these cellular processes. We have characterized the phosphorylation-based regulation of nestin during different stages of the cell cycle in a neuronal progenitor cell line, ST15A. Confocal microscopy of nestin organization and (32)P in vivo labeling studies show that the mitotic reorganization of nestin is accompanied by elevated phosphorylation of nestin. The phosphorylation-induced alterations in nestin organization during mitosis in ST15A cells are associated with partial disassembly of nestin filaments. Comparative in vitro and in vivo phosphorylation studies identified cdc2 as the primary mitotic kinase and Thr(316) as a cdc2-specific phosphorylation site on nestin. We generated a phosphospecific nestin antibody recognizing the phosphorylated form of this site. By using this antibody we observed that nestin shows constitutive phosphorylation at Thr(316), which is increased during mitosis. This study shows that nestin is reorganized during mitosis and that cdc2-mediated phosphorylation is an important regulator of nestin organization and dynamics during mitosis.

In the yeast two-hybrid screening, we have isolated a cDNA clone from a human heart library using Nck Src homology 3 (SH3) domains as bait. The full-length cDNA, which encoded 722 amino acids, was identified as a VIP54-related gene containing an SH3 domain, proline-rich motifs, a serine/threonine-rich region, and a long C-terminal hydrophobic region. We refer to this protein as SPIN90 (SH3 Protein Interacting with Nck, 90 kDa). The amino acid sequence of the SH3 domain has the highest homology with those of Fyn, Yes, and c-Src. SPIN90 was broadly expressed in human tissues; in particular, it was highly expressed in heart, brain, and skeletal muscle, and its expression was developmentally regulated during cardiac myocyte differentiation. SPIN90 is able to bind to the first and third SH3 domains of Nck, in vitro, and is colocalized with Nck at sarcomere Z-discs within cardiac myocytes. Moreover, treatment with antisera raised against SPIN90 disrupted sarcomere structure, suggesting that this protein may play an important role in the maintenance of sarcomere structure and/or in the assembly of myofibrils into sarcomeres.

We previously identified the AF3p21 gene, a novel fusion partner of the MLL gene, in a patient who had developed therapy-related leukemia with t(3;11)(p21;q23). The AF3p21 gene encodes a protein consisting of 722 amino acids, which has an SH3 (Src homology 3) domain, a proline-rich domain, and a bipartite nuclear localization signal. The protein's SH3 domain has high homology with that of FYN. Analysis of the DNA from the patient's leukemic cells revealed that intron 6 of the MLL gene was fused at a point upstream of exon 1 in the AF3p21 gene, and that the der(11) chromosome formed an MLL-AF3p21 fusion transcript in leukemic cells, whereas the der(3) chromosome did not form any fusion transcript. The AF3p21 gene on chromosome band 3p21 is 19 kb long and consists of 13 exons. The size of the mRNA of the AF3p21 gene is approximately 3.5 kb. The AF3p21 gene is widely expressed in normal human tissues including the bone marrow, brain, liver, thymus, lung, and skeletal muscle. Western blot and immunocytochemical analyses showed that AF3p21 protein has an apparent molecular weight of 80 kDa and is localized exclusively in the cell nucleus. These results suggest the possibility that AF3p21 protein plays a role in signal transduction in the nucleus., (Copyright 2001 Wiley-Liss, Inc.)

IgG antifilaggrin autoantibodies (AFA) are the most specific serological markers of rheumatoid arthritis. In epithelial tissues, they recognize citrulline-bearing epitopes present on various molecular forms of (pro)filaggrin. Histological analysis of rheumatoid synovial membranes with an Ab to citrulline showed labeling of interstitial amorphous deposits and mononuclear cells of various types. Immunochemical analysis of exhaustive sequential extracts of the same tissues showed that they contain several deiminated (citrulline containing) proteins. Among them, two proteins, p64--78 and p55--61, present in urea-DTT and guanidine extracts, were shown by immunoblotting to be specifically targeted by AFA. By amino-terminal sequencing the proteins were identified as deiminated forms of the alpha- and beta-chains of fibrin, respectively. Their identity was confirmed using several Abs specific for the A alpha- and/or to the B beta-chain of fibrin(ogen). Moreover, AFA-positive rheumatoid arthritis (RA) sera and purified AFA were highly reactive to the A alpha- and B beta-chains of human fibrinogen only after deimination of the molecules by a peptidylarginine deiminase. Autoantibodies affinity purified from a pool of RA sera onto deiminated fibrinogen were reactive toward all of the epithelial and synovial targets of AFA. This confirmed that the autoantibodies to the deiminated A alpha-and B beta-chains of fibrinogen, the autoantibodies to the synovial proteins p64--78 and p55--61, and, lastly, AFA, constitute largely overlapping autoantibody populations. These results show that deiminated forms of fibrin deposited in the rheumatoid synovial membranes are the major target of AFA. They suggest that autoimmunization against deiminated fibrin is a critical step in RA pathogenesis.

Dystrophin coordinates the assembly of a complex of structural and signaling proteins that are required for normal muscle function. A key component of the dystrophin protein complex is alpha-dystrobrevin, a dystrophin-associated protein whose absence results in neuromuscular junction defects and muscular dystrophy. To gain further insights into the role of alpha-dystrobrevin in skeletal muscle, we used the yeast two-hybrid system to identify a novel alpha-dystrobrevin-binding partner called syncoilin. Syncoilin is a new member of the intermediate filament superfamily and is highly expressed in skeletal and cardiac muscle. In normal skeletal muscle, syncoilin is concentrated at the neuromuscular junction, where it colocalizes and coimmunoprecipitates with alpha-dystrobrevin-1. Expression studies in mammalian cells demonstrate that, while alpha-dystrobrevin and syncoilin associate directly, overexpression of syncoilin does not result in the self-assembly of intermediate filaments. Finally, unlike many components of the dystrophin protein complex, we show that syncoilin expression is up-regulated in dystrophin-deficient muscle. These data suggest that alpha-dystrobrevin provides a link between the dystrophin protein complex and the intermediate filament network at the neuromuscular junction, which may be important for the maintenance and maturation of the synapse.

Sodium butyrate (NaBu) was shown to induce differentiation and apoptosis in the human pancreatic cancer cell line AsPC-1. A suppression subtractive hybridization-based technique was used to identify genes induced by NaBu. A novel cDNA was found to be highly up-regulated in AsPC-1 cells in response to NaBu. The gene expresses a 1.65-kb mRNA encoding a protein with an open reading frame of 422 amino acids. It has an intermediate filament signature sequence and extensive homology to type I keratins. Sequence comparison with known keratins indicated that the gene shares 42-46% amino acid identity and 60-65% similarity within the alpha-helical rod domain. The gene is named K23 (for human type I Keratin 23, KRT23). K23 mRNA was highly induced by NaBu in different pancreatic cancer cells. Trichostatin A (TSA), a potent and specific inhibitor of histone deacetylase, similarly induced K23 mRNA expression. Treatment with either actinomycin D or cycloheximide efficiently blocked the induction of K23 mRNA by NaBu/TSA. These results indicate that K23 mRNA induction by NaBu or TSA is a downstream event of histone hyperacetylation. We also demonstrated that expression of p21(WAF1/CIP1) antisense RNA efficiently blocked the induction of K23 mRNA induced by NaBu. Our results suggest that K23 is a novel member of the acidic keratin family that is induced in pancreatic cancer cells undergoing differentiation by a mechanism involving histone hyperacetylation. p21(WAF1/CIP1) serves as an important mediator during the induction process of K23 by NaBu., (Copyright 2000 Wiley-Liss, Inc.)

Members of the annexin protein family interact with members of the S100 protein family thereby forming heterotetramers in which an S100 homodimer crossbridges two copies of the pertinent annexin. Previous work has shown that S100A1 and S100B bind annexin VI in a Ca(2+)-dependent manner and that annexin VI, but not annexin V, blocks the inhibitory effect of S100A1 and S100B on intermediate filament assembly. We show here that both halves of annexin VI (i.e., the N-terminal half or annexin VI-a and the C-terminal half or annexin VI-b) bind individual S100s on unique sites and that annexin VI-b, but not annexin VI-a, blocks the ability of S100A1 and S100B to inhibit intermediate filament assembly. We also show that the C-terminal extension of S100A1 (and, by analogy, S100B), that was previously demonstrated to be critical for S100A1 and S100B binding to several target proteins including intermediate filament subunits, is not part of the S100 surface implicated in the recognition of annexin VI, annexin VI-a, or annexin VI-b. Evaluation of functional properties with a liposome stability and a calcium influx assay reveals the ability of both S100 proteins to permeabilize the membrane bilayer in a similar fashion like annexins. When tested in combinations with different annexin proteins both S100 proteins mostly lead to a decrease in the calcium influx activity although not all annexin/S100 combinations behave in the same manner. Latter observation supports the hypothesis that the S100-annexin interactions differ mechanistically depending on the particular protein partners.

The RC2 antibody is frequently used to label mouse radial glial cells in all parts of the nervous system where neuronal migration occurs during embryonic and early postnatal life. The antigen recognized by this antibody still needs to be identified. We have characterized further its localization in vivo, its expression and subcellular localization in vitro, as well as its molecular nature. Histologic investigations of whole mouse embryos reveal an equally intense expression of RC2 immunostaining in radial glial cells in brain and spinal cord and in skeletal muscle. In glial cells cultures, the RC2 antibody recognizes an epitope located on the glial cytoskeleton and identified as an intermediate filament associated protein (IFAP) at the ultrastructural level. RC2 immunostaining in those cells is strongly dependent on the presence of a serum-derived activity. Serum-removal causes a decrease of the staining while adding serum back to the cells induces reexpression of RC2 immunoreactivity. By Western blotting, we find that in intermediate filament (IF) preparations obtained from cultured cerebellar glia, the RC2 antibody recognizes a 295-kDa protein whose expression is also dependent on the presence of serum in culture medium. In developing muscle cells, RC2 immunostaining is observed from the myoblast stage and disappears after complete myotube fusion. Both in vivo and in vitro, staining is first seen as a loose capping around myoblasts nuclei and progressively concentrates into Z-disks in association with the muscle IF protein desmin. The RC2 antibody also recognizes a 295-kDa protein band in muscle tissue protein extracts. Thus, the RC2 antibody recognizes a developmentally regulated cytoskeletal protein that is expressed, like other previously identified IFAPs, by cells of the glial and myogenic lineages and whose expression in vitro seems to be controlled by a signaling mechanism known to modulate astroglial morphology., (Copyright 2000 Wiley-Liss, Inc.)

Class V myosins are a ubiquitously expressed family of actin-based molecular motors. Biochemical studies on myosin-Va from chick brain indicate that this myosin is a two-headed motor with multiple calmodulin light chains associated with the regulatory or neck domain of each heavy chain, a feature consistent with the regulatory effects of Ca(2+) on this myosin. In this study, the identity of three additional low molecular weight proteins of 23-,17-, and 10 kDa associated with myosin-Va is established. The 23- and 17-kDa subunits are both members of the myosin-II essential light chain gene family, encoded by the chicken L23 and L17 light chain genes, respectively. The 10-kDa subunit is a protein originally identified as a light chain (DLC8) of flagellar and axonemal dynein. The 10-kDa subunit is associated with the tail domain of myosin-Va., (Copyright 2000 Wiley-Liss, Inc.)

In the stratum granulosum of mammalian epidermis, keratohyalin contains the histidine-rich protein filagrin which determines the aggregation of keratin bundles in terminally differentiating keratinocytes. Among reptiles, the lizard epidermis possesses keratohyalin-like granules during a period preceding molting. Tritiated histidine is taken up at 3-22 h post-injection mainly in keratohyalin-like granules of the clear layer, and less intensely in oberhautchen cells, in pre-keratinizing cells of wound epidermis and in the alpha-layer. No labelling was observed in beta-cells and mesos cells. Single and double immunogold localization of filagrin and keratin, using mammalian antibodies, show that some filagrin-like immunoreactivity is localized in 0.15-0.40 microm diameter roundish keratohyalin-like granules in cells of wound epidermis, and lacunar and clear layers (soft alpha-layers). Pro-filagrin was not immuno-localized in these layers with the mammalian antibodies. The small granules merge with keratin bundles into mature keratinocytes where immunopositive keratin filaments predominate and the filagrin-like immunolabelling rapidly disappears. Little or no labelling is observed in the large keratohyaline-like granules of the clear layer. This may be due to lack of filagrin-like immunoreactivity but may also be due to epitope-masking or chemical degradation of filagrin-like molecules. No immunoreactivity is present in the beta-layer and mesos-layer but the immunolabelling reappears in the maturing alpha-layer and lacunar layer. This study suggests that histidin-rich protein with some filagrin-like immunoreactivity is initially present in those alpha-layers of lizard epidermis where keratohyalin-like granules are present, such as lacunar and clear cells, and that a filagrin-like molecule is degraded or altered in mature keratinocytes.

The Fas/Apo-1/CD95 cell surface receptor belongs to the TNF receptor family of cell death inducing molecules. A number of cytosolic adapter proteins that mediate signal transduction of CD95 have been characterized, but some features of the molecular mechanisms of CD95-induced cell death remain elusive. We describe here a novel protein that can interact with the cytosolic domain of the murine CD95 receptor in a yeast two-hybrid assay. This novel protein was termed Fbf-1 for Fas binding factor and bears no sequence similarity to the known CD95 adapter proteins. Fbf-1 is 1173 aa long and has a theoretical molecular weight of around 130 kDa. The protein is expressed in a wide variety of tissues and is localized in the cytoplasm. Fbf-1 is a very hydrophilic protein, highly conserved between mouse and human and bears a carboxyterminal leucine heptad repeat reminiscent of leucine zipper protein interaction domains. In addition, it shows sequence similarity to trichohyalin and plectin pointing to a function as a structural protein.

Three distinct mRNAs have been shown to be produced by alternative splicing from the unique mouse peripherin gene. They generate three translation products, one major form, Pe-58, and two minor forms, Pe-56 which possess a shorter C-terminal sequence, and Pe-61 in which an additional sequence has been inserted in the central rod domain (Landon et al., 1989, EMBO J. 8, 1719-1726). In this study, the simultaneous occurrence of multiple transcripts in murine nervous tissues and neuroblastoma cell lines was shown by PCR amplification of fragments overlapping the sites of alternative splicing. Recombinant peripherin isoforms were purified from E. coli expressing full-length cDNAs. Rabbit antisera were raised against synthetic peptides mimicking parts of the two C-terminal sequences and of the inserted sequence of Pe-61 and were immunoadsorbed until they became monoreactive. By western blot analysis, the peripherin isoforms were localised in neuroblastoma NB2a cell lysates and detergent insoluble fractions separated by two-dimensional electrophoresis. In addition, each isoform was resolved into several charge variants. At the cellular level, each antibody decorated the filament array of the NB2a cells, suggesting the participation of the minor peripherin isoforms in the intermediate filament network.

Two novel cytoplasmic intermediate filament (IF) proteins (C and D) from the tunicate (urochordate) Styela are characterised as putative keratin orthologs. The coexpression of C and D in all epidermal cells and the obligatory heteropolymeric IF assembly of the recombinant proteins argue for keratin orthologs, but the sequences do not directly reveal which protein behaves as a keratin I or II ortholog. This problem is solved by the finding that keratin 8, a type II keratin from man or Xenopus, forms chimeric IF when mixed with Styela D. Mutant proteins of Styela D and keratin 8 with a single cysteine in equivalent positions show that these chimeric IF are, like vertebrate keratin filaments, based on the hetero coiled coil. We propose that Styela D retains, in spite of its strong sequence drift, important molecular features of type I keratins. By inference Styela C reflects a type II ortholog. We discuss that type I to III IF proteins are expressed along the chordate branch of metazoa.

Plectin is a high-molecular-weight cytoskeleton-associated protein that was initially identified in intermediate filament (IF)-enriched fractions of rat C6 glioma cells. At the cellular level, plectin has been found to associate with IF networks and IF-associated structures that are involved in cell-cell and cell-substrate adhesions. IFAP300 is an IF-associated protein that was initially identified in hamster cells by a monoclonal antibody directed against a high molecular weight protein present in IF-enriched cytoskeletal preparations. Plectin and IFAP300 display similar distribution patterns within cells as determined by immunofluorescence. Based upon this and the finding that their biochemical properties are similar, it has been suggested that they may actually be orthologous proteins. In this paper we demonstrate that this is the case. Cloning and sequencing of most of the hamster plectin cDNA demonstrates that plectin is found in hamster cells and that its sequence is highly conserved between species. Using immunological cross-reactivity, epitope mapping, and immunoelectron microscopy, we show that IFAP300 is actually the hamster ortholog of plectin., (Copyright 2000 Academic Press.)

The amino acid residues responsible for stable binding of nucleic acids by the intermediate filament (IF) subunit protein vimentin were identified by a combination of enyzmatic and chemical ladder sequencing of photo-cross-linked vimentin-oligodeoxyribonucleotide complexes and analysis by MALDI-TOF mass spectrometry. Three tryptic peptides of vimentin (vim(28)(-)(35), vim(36)(-)(49), and vim(50)(-)(63)) were found to be cross-linked to oligo(dG.BrdU)(12). dG.3'-FITC. From a methodological standpoint, it was necessary to remove the bulk of the bound oligonucleotide by digestion with nuclease P1 to get reproducible spectra for most of the peptides studied. Additionally, removal of the phosphate group of the residually bound dUMP or modification of the amino terminus of the peptide-oligonucleotide complexes with dimethylaminoazobenzene isothiocyanate dramatically improved the quality of the MALDI-TOF spectra obtained, particularly for the vim(28)(-)(35) peptide. A single Tyr residue within each of these peptides (Tyr(29), Tyr(37), and Tyr(52)) was unequivocally demonstrated to be the unique site of cross-linking in each peptide. These three Tyr residues are contained within the two beta-ladder DNA-binding wings proposed for the middle of the vimentin non-alpha-helical head domain. The experimental approach described should be generally applicable to the study of protein-nucleic acid interactions and is currently being employed to characterize the DNA-binding sites of several other IF subunit proteins.

Nearly all intermediate filament proteins exhibit a highly conserved amino acid motif (YRKLLEGEE) at the C-terminal end of their central alpha-helical rod domain. We have analyzed its contribution to the various stages of assembly by using truncated forms of Xenopus vimentin and mouse desmin, VimIAT and DesIAT, which terminate exactly before this motif, by comparing them with the wild-type and tailless proteins. It is surprising that in buffers of low ionic strength and high pH where the full-length proteins form tetramers, both VimIAT and DesIAT associated into various high molecular weight complexes. After initiation of assembly, both VimIAT and DesIAT aggregated into unit-length-type filaments, which rapidly longitudinally annealed to yield filaments of around 20 nm in diameter. Mass measurements by scanning transmission electron microscopy revealed that both VimIAT and DesIAT filaments contained considerably more subunits per cross-section than standard intermediate filaments. This indicated that the YRKLLEGEE-motif is crucial for the formation of authentic tetrameric complexes and also for the control of filament width, rather than elongation, during assembly. To determine the structure of the YRKLLEGEE domain, we grew crystals of peptides containing the last 28 amino acid residues of coil 2B, chimerically fused at its amino-terminal end to the 31 amino acid-long leucine zipper domain of the yeast transcription factor GCN4 to facilitate appropriate coiled-coil formation. The atomic structure shows that starting from Tyr400 the two helices gradually separate and that the coiled coil terminates with residue Glu405 while the downstream residues fold away from the coiled-coil axis., (Copyright 2000 Academic Press.)

Juvenile-onset cataracts are distinguished from congenital cataracts by the initial clarity of the lens at birth and the gradual development of lens opacity in the second and third decades of life. Genomewide linkage analysis in a multigenerational pedigree, segregating for autosomal dominant juvenile-onset cataracts, identified a locus in chromosome region 3q21.2-q22.3. Because of the proximity of the gene coding for lens beaded filament structural protein-2 (BFSP2) to this locus, we screened for mutations in the coding sequence of BFSP2. We observed a unique C-->T transition, one that was not observed in 200 normal chromosomes. We predicted that this led to a nonconservative R287W substitution in exon 4 that cosegregated with cataracts. This mutation alters an evolutionarily conserved arginine residue in the central rod domain of the intermediate filament. On consideration of the proposed function of BFSP2 in the lens cytoskeleton, it is likely that this alteration is the cause of cataracts in the members of the family we studied. This is the first example of a mutation in a noncrystallin structural gene that leads to a juvenile-onset, progressive cataract.

Congenital cataracts are a common major abnormality of the eye that frequently cause blindness in infants. At least one-third of all cases are familial; autosomal-dominant congenital cataract appears to be the most-common familial form in the Western world. Elsewhere, in family ADCC-3, we mapped an autosomal-dominant cataract gene to chromosome 3q21-q22, near the gene that encodes a lens-specific beaded filament protein gene, BFSP2. By sequencing the coding regions of BFSP2, we found that a deletion mutation, DeltaE233, is associated with cataracts in this family. This is the first report of an inherited cataract that is caused by a mutation in a cytoskeletal protein.

The molecular dynamics in the vicinity of sulfhydryl groups of stratum corneum (SC) proteins has been studied by electron paramagnetic resonance (EPR) spectroscopy of maleimide spin labels covalently bound to the proteins. The total amount of bound maleimide was around 4 nmol per mg of SC. We have interpreted the coexistence of two spectral components in the EPR spectra by a two-state model with a fraction of label hydrogen bonded to proteins and another fraction exposed to the aqueous environment. We showed that the relative populations among these two states, determined by spectral simulation, are in thermodynamic equilibrium. The calculated energetic gain for the nitroxide to form hydrogen bond with SC proteins rather than to be dissolved in the buffer was approximately 12 kcal/mol in the temperature range of 2-30 degrees C and approximately 5 kcal/mol in the range of 30-86 degrees C. Temperature profiles of other EPR parameters related to the rotational diffusion of the probe also showed changes in the temperature interval of 26-42 degrees C, suggesting alterations in the vibration modes of SC proteins which are sensitive to higher motional freedom above 26-42 degrees C. We also compared samples of intact and lipid-depleted SC and we found that the delipidization process does not alter significantly the backbone mobility in the SH group regions, but the data suggest that the protein cavity is more open in the case of the delipidized samples. These results contribute to the understanding of the protein participation in the barrier function of SC, and can be useful to improve the spectral analysis of site-directed spin labeling, particularly for a more quantitative description of the dynamic modes of the nitroxide side chains.

Peripherin/Rds is a tetraspanning membrane protein that has been implicated in photoreceptor outer segment morphogenesis and inherited retinal degenerative diseases. Together with the structurally related protein, Rom-1, it forms a complex along the rims of rod and cone disc membranes. We have compared the oligomeric structure of these proteins from nonreduced and dithiothreitol reduced membranes by velocity sedimentation, SDS-gel electrophoresis, immunoaffinity chromatography, and chemical cross-linking. Under reducing conditions peripherin/Rds and Rom-1 existed as homomeric and heteromeric core complexes devoid of intermolecular disulfide bonds. Under nonreducing conditions core complexes associated through intermolecular disulfide bonds to form oligomers. One intermediate-size oligomer contained monomers and disulfide-linked dimers of peripherin/Rds and Rom-1, while larger oligomers consisted only of disulfide-linked peripherin/Rds dimers when analyzed on nonreducing SDS gels. Consistent with this result, disc membranes contained twice as much peripherin/Rds as Rom-1. Peripherin/Rds individually expressed in COS-1 cells also formed disulfide-linked oligomers bridged through Cys-150 residues, whereas Rom-1 showed little tendency to form oligomers. These results indicate that peripherin/Rds and Rom-1 associate noncovalently to form multisubunit core complexes. Peripherin/Rds containing core complexes interact through specific intermolecular disulfide bonds to form oligomers which may play a crucial role in photoreceptor disc morphogenesis and retinal degenerative diseases.

Photoreceptor peripherin/rds promotes membrane fusion, through a putative fusion domain located within the C-terminus (Boesze-Battaglia et al., Biochemistry 37 (1998) 9477-9487). A peptide analogue to this region, PP-5, competitively inhibits peripherin/rds mediated fusion in a cell free assay system. To characterize how this region is involved in the fusion process we investigated two of the individual steps in membrane fusion, membrane adhesion and membrane destabilization inferred from depolarization studies. Membrane depolarization was measured as the collapse of a valinomycin induced K(+) diffusion potential in model membranes, using a potential sensitive fluorescent probe, diS-C(2)-5. PP-5 induced membrane depolarization in a concentration dependent manner. PP-5 has been shown by Fourier transform infrared spectroscopy to be an amphiphilic alpha-helix. Therefore, the requirement for an amphiphilic alpha-helix to promote depolarization was tested using two mutant peptides designed to disrupt either the amphiphilic nature of PP-5 (PP-5AB) or the alpha-helical structure (PP-5HB). PP-5AB inhibited PP-5 induced depolarization when added in an equimolar ratio to PP-5. Neither mutant peptide alone or in combination with PP-5 had any effect on calcium dependent vesicle aggregation. Using non-denaturing gel electrophoresis and size exclusion chromatography techniques PP-5 was shown to form a tetrameric complex. Equimolar mixtures of PP-5 and PP-5AB formed a heterotetramer which was unable to promote membrane depolarization. The hypothesis that PP-5 tetramers promote membrane depolarization is consistent with the calculated Hill coefficient of 3.725, determined from a Hill analysis of the depolarization data.