Antiparallel orientation of the two double-stranded coiled-coils in the tetrameric protofilament unit of intermediate filaments

The chymotryptically excised middle domain of desmin slightly exceeds in length the structurally conserved alpha-helical middle region documented in all intermediate filament proteins by amino acid sequence data. This rod domain is a protofilament derivative with a tetrameric organization, thus indicating the presence of two double-stranded coiled-coil units. We now show by immunoelectron microscopy that Fab fragments of a desmin-specific monoclonal antibody mixed with the rod lead to dumb-bell-shaped structures. The tagging of both ends together with the length of the rod (48 nm) argues for an antiparallel orientation of the two coiled-coils without a major stagger. This information combined with the lateral 21 nm periodicity of the intermediate filament observed by us and others leads to a structural hypothesis similar to those entertained from X-ray data on wool alpha-keratins, although here an antiparallel tetrameric unit of some 60 to 66 nm is invoked, which has never been isolated. The structure that we discuss allows for the existence of both the particles, and the antibody experiment strongly supports the antiparallel orientation postulated in both approaches. The tube-like filament structure proposed for the intermediate filament agrees with recent mass per unit length measurements and allows for two minor classes of intermediate filaments with different values in this property as also found experimentally.     

Desmin and Vimentin Intermediate Filament Networks: Their Viscoelastic Properties Investigated by Mechanical Rheometry

We have investigated the viscoelastic properties of the cytoplasmic intermediate filament (IF) proteins desmin and vimentin. Mechanical measurements were supported by time-dependent electron microscopy studies of the assembly process under similar conditions. Network formation starts within 2 min, but it takes more than 30 min until equilibrium mechanical network strength is reached. Filament bundling is more pronounced for desmin than for vimentin. Desmin filaments (persistence length l_p ≈ 900 nm) are stiffer than vimentin filaments (l_p ≈ 400 nm), but both IFs are much more flexible than microfilaments. The concentration dependence of the plateau modulus G₀ ∼ c^α is much weaker than predicted theoretically for networks of semiflexible filaments. This is more pronounced for vimentin (α = 0.47) than for desmin (α = 0.70). Both networks exhibit strain stiffening at large shear deformations. At the transition from linear to nonlinear viscoelastic response, only desmin shows characteristics of nonaffine network deformation. Strain stiffening and the maximum modulus occur at strain amplitudes about an order of magnitude larger than those for microfilaments. This is probably attributable to axial slippage within the tetramer building blocks of the IFs. Network deformation beyond a critical strain γ_max results in irreversible damage. Strain stiffening sets in at lower concentrations, is more pronounced, and is less sensitive to ionic strength for desmin than for vimentin. Hence, desmin exhibits strain stiffening even at low-salt concentrations, which is not observed for vimentin, and we conclude that the strength of electrostatic repulsion compared to the strength of attractive interactions forming the network junctions is significantly weaker for desmin than for vimentin filaments. These findings indicate that both IFs exhibit distinct mechanical properties that are adapted to their respective cellular surroundings [i.e., myocytes (desmin) and fibroblasts (vimentin)].     

Near-UV Circular Dichroism Reveals Structural Transitions of Vimentin Subunits during Intermediate Filament Assembly

In vitro assembly of vimentin intermediate filaments (IFs) proceeds from soluble, reconstituted tetrameric complexes to mature filaments in three distinct stages: (1) within the first seconds after initiation of assembly, tetramers laterally associate into unit-length filaments (ULFs), on average 17 nm wide; (2) for the next few minutes, ULFs grow by longitudinal annealing into short, immature filaments; (3) almost concomitant with elongation, these immature filaments begin to radially compact, yielding ∼ 11-nm-wide IFs at around 15 min. The near-UV CD signal of soluble tetramers exhibits two main peaks at 285 and 278 nm, which do not change during ULF formation. In contrast, the CD signal of mature IFs exhibits two major changes: (1) the 278-nm band, denoting the transition of the tyrosines from the ground state to the first vibrational mode of the excited state, is lost; (2) a red-shifted band appears at 291 nm, indicating the emergence of a new electronic species. These changes take place independently and at different time scales. The 278-nm signal disappears within the first minute of assembly, compatible with increased rigidity of the tyrosines during elongation of the ULFs. The rise of the 291-nm band has a lifetime of ∼ 13 min and denotes the generation of phenolates by deprotonation of the tyrosines' hydroxyl group after they relocalize into a negatively charged environment. The appearance of such tyrosine-binding “pockets” in the assembling filaments highlights an essential part of the molecular rearrangements characterizing the later stages of the assembly process, including the radial compaction.     

Immunochemical comparison of prekeratin and alcoholic hyalin intermediate filaments

Alcoholic hyalin is an hepatocellular aggregate composed of filaments apparently related to the prekeratin intermediate filament subclass. The relationship between these two filament preparations was determined immunochemically using guinea pig antisera derived against alcoholic hyalin, prekeratin, and major prekeratin polypeptides. Immunocrossreactivities were determined using sensitive solid-phase enzyme-immunoassays. These assays indicated that antisera derived against a given filament preparation reacted 10–1000 times better with that preparation than with the other system. The nature of crossreactive meterial was determined using antisera derived against the larger prekeratin polypeptides (M_r 61,000 and 51,000). When tested against these two antisera, alcoholic hyalin appeared to react better with the serum derived against the larger prekeratin component. Moreover, anti-alcoholic hyalin antiserum bound four to five times better to the 61,000 dalton component than to the 51,000 dalton polypeptide in the enzyme-immunoassay. Our results indicate that antigenic determinants related to prekeratin can be detected in alcoholic hyalin, but that these determinants are present in relatively low concentrations in purified alcoholic hyalin. In addition, it appears that the relative concentrations of prekeratin components in alcoholic hyalin do not reflect those in purified prekeratin.     

Intermediate filaments: a historical perspective

Intracellular protein filaments intermediate in size between actin microfilaments and microtubules are composed of a surprising variety of tissue specific proteins commonly interconnected with other filamentous systems for mechanical stability and decorated by a variety of proteins that provide specialized functions. The sequence conservation of the coiled-coil, alpha-helical structure responsible for polymerization into individual 10 nm filaments defines the classification of intermediate filament proteins into a large gene family. Individual filaments further assemble into bundles and branched cytoskeletons visible in the light microscope. However, it is the diversity of the variable terminal domains that likely contributes most to different functions. The search for the functions of intermediate filament proteins has led to discoveries of roles in diseases of the skin, heart, muscle, liver, brain, adipose tissues and even premature aging. The diversity of uses of intermediate filaments as structural elements and scaffolds for organizing the distribution of decorating molecules contrasts with other cytoskeletal elements. This review is an attempt to provide some recollection of how such a diverse field emerged and changed over about 30 years.     

甲醇酵母Pichia pastoris高水平表达有活性的辣根过氧化物酶

表达有活性的辣根过氧化物酶（ＨＲＰ） 不仅可以深入揭示ＨＲＰ 结构与功能及其生理作用规律, 而且为ＨＲＰ的广泛需要提供新的来源． 为了在甲醇酵母Ｐ． ｐａｓｔｏｒｉｓ 中成功表达, 将编码ＨＲＰＣ成熟肽的ｃＤＮＡ 构建到ｐＰＩＣ９ 上, 再转化到Ｐ． ｐａｓｔｏｒｉｓ 中, 筛选到了分泌表达非糖基化ＨＲＰ 和高糖基化ＨＲＰ（ 分子质量超过１００ ｋｕ） 两种主要产物的重组细胞株． 优化表达条件, 目标产物在摇瓶发酵液中高效表达, 可达４～６ ｇ／Ｌ． 并且直接从发酵液中可获得具有活性的高糖基化ＨＲＰ, 每毫升发酵液中酶活力约有２ Ｕ, 经初步的纯化ＨＲＰ具有最大吸收峰４０３ ｎｍ ．     

Exploring the mechanical properties of single vimentin intermediate filaments by atomic force microscopy

Intermediate filaments (IFs), together with actin filaments and microtubules, compose the cytoskeleton. Among other functions, IFs impart mechanical stability to cells when exposed to mechanical stress and act as a support when the other cytoskeletal filaments cannot keep the structural integrity of the cells. Here we present a study on the bending properties of single vimentin IFs in which we used an atomic force microscopy (AFM) tip to elastically deform single filaments hanging over a porous membrane. We obtained a value for the bending modulus of non-stabilized IFs between 300 MPa and 400 MPa. Our results together with previous ones suggest that IFs present axial sliding between their constitutive building blocks and therefore have a bending modulus that depends on the filament length. Measurements of glutaraldehyde-stabilized filaments were also performed to reduce the axial sliding between subunits and therefore provide a lower limit estimate of the Young's modulus of the filaments. The results show an increment of two to three times in the bending modulus for the stabilized IFs with respect to the non-stabilized ones, suggesting that the Young's modulus of vimentin IFs should be around 900 MPa or higher.     

Assembly characteristics of plant keratin intermediate filaments in vitro

After selective extraction and purification, plant keratin intermediate filaments were reassembled in vitro. Scanning tunneling microscope (STM) and transmission electron microscope (TEM) micrographs showed that acidic keratins and basic keratins can assemble into dimers and further into 10 nm filaments in vitro. In higher magnification images, it can be seen that fully assembled plant keratin intermediate filaments consist of several thinner filaments of 3 nm in diameter, which indicates the formation of protofilaments in the assembly processes. One of the explicit features of plant keratin intermediate filaments is a 24—25 nm periodic structural repeat alone the axis of beth the 10 nm filaments and protofilaments. The periodic repeat is one of the fundamental characteristic of all intermediate filaments, and demonstrates the half staggered arrangement of keratin molecules within the filaments.     

Severe Myopathy Mutations Modify the Nanomechanics of Desmin Intermediate Filaments

Mutations in the intermediate filament (IF) protein desmin cause severe forms of myofibrillar myopathy characterized by partial aggregation of the extrasarcomeric desmin cytoskeleton and structural disorganization of myofibrils. In contrast to prior expectations, we showed that some of the known disease-causing mutations, such as DesA360P, DesQ389P and DesD399Y, are assembly-competent and do allow formation of bona fide IFs in vitro and in vivo. We also previously demonstrated that atomic force microscopy can be employed to measure the tensile properties of single desmin IFs. Using the same approach on filaments formed by the aforementioned mutant desmins, we now observed two different nanomechanical behaviors: DesA360P exhibited tensile properties similar to that of wild-type desmin IFs, whereas DesQ389P and DesD399Y exhibited local variations in their tensile properties along the filament length. Based on these findings, we hypothesize that DesQ389P and DesD399Y may cause muscle disease by altering the specific biophysical properties of the desmin filaments, thereby compromising both its mechanosensing and mechanotransduction ability.     

The Detection of Cyanobacterial Cells by a Non-Fluorescent in situ Hybridization Method

Seven cyanobacterial strains (Anabaena macrospora NIER10016, Oscillatoria sp. NIER10042, Microcystis aeruginosa NIER10015, M. ichtyoblabe NIER10025 and NIER10040, M. novacekii NIER10029, and M. wesenbergii NIER10068) were tested by a nonfluorescent in situ hybridization method using two specific horseradish peroxidase–labeled oligonucleotide probes and two chromogenic substrates. This approach was shown to be appropriate for the analysis of natural samples.     

Colocalization of single ribosomes with intermediate filaments in puromycin-treated and serum-starved mouse embryo fibroblasts*

Previous experiments have revealed a relatively weak electrostatic binding capacity of in vitro reconstituted intermediate filaments (IFs) as well as of natural IFs of whole cell mount preparations for purified ribosomal particles of mammalian origin. In order to demonstrate that such associations also occur in vivo, intact cells were subjected to double immunofluorescence microscopy using antibodies directed against vimentin and ribosomal protein S17. Since in proliferating cells the majority of the ribosomal particles are assembled into polyribosomes and these are to a great extent associated with microfilaments, in vitro cultured mouse embryo skin fibroblasts (MSF cells) were treated with puromycin to allow the formation of single ribosomes. Employing confocal laser scanning microscopy, the ribosomes were detected in colocalization with vimentin IFs. Disassembly of polyribosomes was also achieved by serum starvation of cultured cells. In this case, MSF cells of a low passage attained an extended and flattened appearance with the vimentin IFs being directly associated with the cell nuclei, radiating into the peripheral areas of the cells or showing a stress fiber-like distribution. In both cases, considerable quantities of ribosomal material were seen in close neighborhood to vimentin IFs. Frequently, these ribosome-IF associations were coaligned with microtubules and they also surrounded myosin I-decorated stress fibers. Double labeling with the vital, RNA-specific fluorochrome SYTO 14 produced a fluorescence pattern largely super-imposable on that of ribosomal protein S17. Treatment of the starved cells with either demecolcine or cytochalasin D had an only moderately disturbing effect on vimentin IF distribution and the ribosomes stayed in contact with the vimentin IFs. On the basis of these results, it is conceivable that IFs play a role in the storage of ribonucleoprotein particles in general and non-translating ribosomes in particular in the cytoplasm of animal cells. In addition, the often seen coalignment of IFs with microtubules and microfilaments might serve facilitated and directional transport of ribonucleoprotein particles from the nucleus to peripheral areas of the cell.     

Effect of surfactants on peroxidase activity: IV. Effect of milk lipids and fatty acid salts

The effect of surfactants, lipids and fatty acid salts isolated from cow milk on the activity of hemecontaining horseradish peroxidase in solution was studied. As the surfactant concentration increases, the rate of the enzymic reaction successively decreases, increases, and again decreases, down to zero in the case of the fatty acid salts. The initial deceleration of the reaction rate results from the enzyme inhibition. The subsequent increase is caused by an improved accessibility for the substrate and the enhanced activity of the catalytic site of the enzyme due to its immobilization in the surfactant aggregates. A shielding of the protein by these aggregates can explain the secondary deceleration of the enzymic reaction rate. The general character of the dependence is similar and does not depend on the surfactant structure for a series of fatty acid salts and phospholipids; however, it is quite different in the case of cholesterol and sphingomyelin. For communication III, see [1].     

ACTH-induced internalization of plasma membrane in xanthophores of the goldfish, Carassius auratus L.

The ACTH-induced pigment dispersion in primary cultures and in suspensions of xanthophores of the goldfish, $\text{Carassius}\text{auratus}$ L., has been shown to include the formation of rosette-like membranous structures and plasma membrane invaginations (pits). The rosettes and pits are probably similar structures sectioned at different angles. Horseradish peroxidase studies demonstrate that these structures are eventually converted into small spherical vesicles and long smooth elements, similar in appearance to spherical and “tubular” endoplasmic reticulum, respectively. In addition, these studies show that once vesicles are formed they are no longer continuous to the outside of the cell.     

Modulation of the catalytic activity of free and immobilized peroxidase by extremely low frequency electromagnetic fields: dependence on frequency

A study of the influence of electromagnetic fields (EMF) of various frequencies, from 50 up to 400 Hz, on the catalytic activity of soluble and insoluble horseradish peroxidase (POD) was carried out. To simulate the conditions in which the enzyme operates in vivo, the POD was immobilized by entrapment on a gelatin membrane or by covalent attachment on a nylon graft membrane. The rate of inactivation of the soluble POD was found to exhibit positive and negative interactions with the 1 mT applied magnetic field, with an optimum positive effect at 130 Hz. The immobilized PODs, on the contrary, do not exhibit negative interactions, but show a maximum positive interaction at 150 Hz when entrapped and at 170 Hz when covalently attached. At 50 Hz and at frequencies higher than 250 Hz no effects were observed with insoluble POD. The optimum frequency of positive interaction between the EMF and the catalytic activity of the insoluble enzymes is shifted with respect to that of the soluble enzymes towards higher frequencies, the size of the shifts being dependent on the intensity of the physical forces involved in the immobilization process.     

The Intermediate Filament Protein Peripherin Is the Specific Interaction Partner of Mouse BPAG1-n (Dystonin) in Neurons

The dystonia musculorum (dt) mouse suffers from severe degeneration of primary sensory neurons. The mutated gene product is named dystonin and is identical to the neuronal isoform of bullous pemphigoid antigen 1 (BPAG1-n). BPAG1-n contains an actin-binding domain at its NH₂ terminus and a putative intermediate filament-binding domain at its COOH terminus. Because the degenerating sensory neurons of dt mice display abnormal accumulations of intermediate filaments in the axons, BPAG1-n has been postulated to organize the neuronal cytoskeleton by interacting with both the neurofilament triplet proteins (NFTPs) and microfilaments. In this paper we show by a variety of methods that the COOH-terminal tail domain of mouse BPAG1 interacts specifically with peripherin, but in contrast to a previous study (Yang, Y., J. Dowling, Q.C. Yu, P. Kouklis, D.W. Cleveland, and E. Fuchs. 1996. Cell. 86:655–665), mouse BPAG1 fails to associate with full-length NFTPs. The tail domains interfered with the association of the NFTPs with BPAG1. In dt mice, peripherin is present in axonal swellings of degenerating sensory neurons in the dorsal root ganglia and is downregulated even in other neural regions, which have no obvious signs of pathology. Since peripherin and BPAG1-n also display similar expression patterns in the nervous system, we suggest that peripherin is the specific interaction partner of BPAG1-n in vivo.     

Intermediate filaments: analysis of filamentous aggregates induced by griseofulvin, an antitubulin agent

Mice fed griseofulvin, an antibiotic with antimicrotubular activity, formed hepatocellular aggregates of intermediate filaments, which resembled those associated with human alcoholic liver disease. These aggregates, termed Mallory bodies, were isolated from both human and mouse liver and the composition of these structures compared. Electrophoretic analysis indicated that the mouse filaments were composed of four major polypeptides (51,000, 47,000, 37,000, and 36,000 daltons). Human Mallory bodies possessed a similar number of components but of different molecular weights (56,000, 51,000, 50,000, and 38,000 daltons). Guinea pig antisera prepared against both whole human Mallory bodies and the major human polypeptide (56,000 daltons) crossreacted with mouse Mallory body material in both immunochemical and immunocytochemical systems. Our findings suggest that the two filament systems possess similar biochemical and immunological properties.     

Cytokeratin 18-mediated disorganization of intermediate filaments is induced by degradation of plectin in human liver cells

Plectin is a cross-linking protein that organizes the cytoskeleton into a stable meshwork that helps maintain the uniform size and shape of cells. As cells of hepatocellular carcinoma are morphologically different from healthy human hepatocytes, we hypothesized that plectin deficiency and cytoskeletal disorganization underlies this pleomorphic transformation. To test this hypothesis we induced apoptosis as the most accessible pathway for creating plectin deficiency status in vivo. We analyzed expression levels and organization of plectin and other cytoskeletal elements, including intermediate filaments, microfilaments, and microtubules, after staurosporine-induced apoptosis in human Chang liver cells. The results revealed the expression of plectin and cytokeratin 18 were downregulated in hepatocellular carcinoma tissues in vivo. The expression of actin and tubulin, however, were not altered. In vitro analysis indicated that plectin and cytokeratin 18 were cleaved following staurosporine-treatment of human Chang liver cells. Time course experiments revealed that plectin was cleaved 2 h earlier than cytokeratin 18. The organization of plectin and cytokeratin 18 networks collapsed after staurosporine-treatment. Conclusively, degradation of plectin induced by staurosporine-treatment in liver cells resulted in cytoskeleton disruption and induced morphological changes in these cells by affecting the expression and organization of cytokeratin 18.     

Ectopic expression of desmin in the epidermis of transgenic mice permits development of a normal epidermis

Cell architecture is largely based on the interaction of cytoskeletal proteins, which include intermediate filaments (IF), microfilaments, microtubules, as well as their type-specific membrane-attachment structures and associated proteins. In order to further our understanding of IF proteins and to address the fundamental issue whether different IF perform unique functions in different tissues, we expressed a desmin transgene in the basal epidermis of mice. Ectopic expression of desmin led to the formation of an additional, keratin-independent IF cytoskeleton and did not interfere with the keratin-desmosome interaction. We show that ectopic expression of a type III IF protein in basal keratinocytes did not interfere with the normal epidermal architecture and the program of terminal differentiation. This demonstrated that keratinocytes suffered no obvious detrimental effects from extra desmin filaments in their cytoplasm. In addition, we asked whether stable expression of desmin could rescue K5 null mice, which served as a model for severe EBS. Transgenic mice ectopically expressing desmin in the basal layer were mated with K5 heterozygous deficient animals to generate desmin rescue mice and analysed. In summary, our study support the notion that the different IF like desmin or keratins composing a IF network in vivo are central to cytoskeletal architecture and design in cells.     

甲醇酵母Pichia pastoris高水平表达有活性的辣根过氧化物酶

表达有活性的辣根过氧化物酶(HRP)不仅可以深入揭示HRP结构与功能及其生理作用规律,而且为HRP的广泛需要提供新的来源.为了在甲醇酵母P.pastoris中成功表达,将编码HRP-C成熟肽的cDNA构建到pPIC9上,再转化到P.pastoris中,筛选到了分泌表达非糖基化HRP和高糖基化HRP(分子质量超过100 ku)两种主要产物的重组细胞株.优化表达条件,目标产物在摇瓶发酵液中高效表达,可达4～6 g/L.并且直接从发酵液中可获得具有活性的高糖基化HRP,每毫升发酵液中酶活力约有2 U,经初步的纯化HRP具有最大吸收峰403 nm.     

Interaction of aromatic donor molecules with horseradish peroxidase: identification of the binding site and role of heme iron in the binding and activity

The interaction of aromatic substrates with horseradish peroxidase (HRP) was studied. Chemical modification of HRP was performed using diethylpyrocarbonate (DEPC) and for the first time the amino acid involved in binding with these substrates has been identified. The kinetic parameters for this interaction have been calculated and the role of heme iron in the oxidation of aromatic substrates by HRP has been discussed.