Robert L. Shoeman

Intermediate filament proteins: cytoskeletal elements with gene-regulatory function?

Publisher Summary Intermediate filament (IFs) probably represents one of the last functionally important macromolecular protein assemblies of the eukaryotic cell whose biological role has not yet been unveiled. This chapter discusses the cellular function of IFs, which is based on some characteristic reactivities of their constituent protein subunits with nuclear components in vitro. The structure and function of the IF proteins and their oligo- and polymeric forms are compared with a variety of gene-regulatory protein factors and nuclear matrix elements and shown to have many features in common with these compounds. IF proteins are a class of regulatory DNA binding factors which, in accordance with their developmentally and tissue-specifically regulated expression, may play an important role in cell differentiation as one of their major biological functions.

Isolation of SDS−stable complexes of the intermediate filament protein vimentin with repetitive, mobile, nuclear matrix attachment region, and mitochondrial DNA sequence elements from cultured mouse and human fibroblasts

Crosslinkage of vimentin to DNA in mouse L929 cells by formaldehyde and isolation of SDS-stable DNA-vimentin complexes from normal L929 cells and mouse and human embryo fibroblasts indicated close spatial relations between these components in the intact cell. The adducts, obtained by immunoprecipitation with anti-vimentin antibody, contained substantial quantities, not only of repetitive and mobile sequence elements such as centromeric satellite DNA, telomere DNA, microsatellites and minisatellites, long and short interspersed nucleotide elements, and retroposons, but also of mitochondrial (mt) DNA. Because the SDS-stable complexes could be isolated with distinctly higher yields from oxidatively stressed, senescent fibroblasts and were dissociated by boiling, they possibly arose from accidental condensation reactions mediated by unsaturated and dialdehydes, products of free radical-induced lipid peroxidation. They can therefore be considered vestiges of a general interaction of vimentin with cellular DNA. The sequence patterns of their DNA fragments were similar to those of extrachromosomal circular and linear DNA, including retroviral elements, markers and enhancers of genomic instability that also occur in the cytoplasm and are able to transport vimentin into the nucleus. Many of the fragments were also remarkably similar to AT-rich nuclear matrix attachment regions (MARs) in that they contained, in addition to various mobile elements, a palette of typical MAR motifs. With its tendency to multimerize and to interact with single-stranded and supercoiled DNA, vimentin thus behaves like a nuclear matrix protein and may as such participate in a variety of nuclear matrix-associated processes such as replication, recombination, repair, and transcription of DNA. These activities seem to be extendible to the mitochondrial compartment, as vimentin was also crosslinked to mtDNA, preferentially to its D-loop and hypervariable main control region. These sites are prone to point and deletion mutations and, like nuclear MARs, are associated with the cyto-karyomatrix. Moreover, as a developmentally regulated and tissue-specific cyto-karyomatrix protein, vimentin may contribute to the organization of chromatin, including centromeric and telomeric heterochromatin at the nuclear periphery, with all its consequences for genomic activities during embryogenesis and in adulthood of vertebrates. However, because of its high affinity for hypervariable, recombinogenic DNA sequences, vimentin is proposed to play a major role in both the preservation and the evolution of the nuclear and mitochondrial genome.

Degradation of cytoskeletal proteins by the human immunodeficiency virus type 1 protease

Triton X-100-extracted human skin fibroblasts were exposed to human immunodeficiency virus type 1 protease and analysed by 2D-gel electrophoresis and immunofluorescence microscopy. Vimentin, two of the tropomyosin isoforms, a protein with M(r) approximately 90,000 and a protein with M(r) approximately 200,000 were found to be degraded. Structurally, this was accompanied by the disintegration of the vimentin filament network and the disappearance of the microfilament network. In contrast to our in vivo observations (Höner et al., 1991), prominent stress fibers and chromatin structure seemed to be rather resistant to the action of this protease.

Potential role of the viral protease in human immunodeficiency virus type 1 associated pathogenesis

Infection with the human immunodeficiency virus type 1 (HIV-1) results in a variety of pathological changes culminating in the acquired immune deficiency syndrome (AIDS). While most of these changes can readily be accounted for either by direct effects of HIV-1 on the immune system or by indirect effects of secondary infectious agents as a result of faulty immune surveillance, the direct cause for a number of disease states, including some neuropathies, myopathies, nephropathy, thrombocytopenia, wasting syndromes and increased incidence of cancers (primarily lymphoma) has remained an enigma. We have recently shown that the HIV-1 protease, a viral encoded enzyme necessary for virus maturation and infectivity, can cleave a variety of host cell cytoskeletal proteins in vitro. Potential substrates for the HIV-1 protease are found in all of the cell types affected in these unexplained diseases. Recent proposals suggest that elements of the cytoskeleton may play an important role in the regulation of large scale genetic regulation. We propose that some of the degenerative changes associated with infection by HIV-1 are a direct consequence of cleavage of host cell cytoskeletal proteins, which in turn may be responsible for the increased incidence of cancer in HIV-1 infected individuals as a result of the perturbation of the regulation of gene expression by cytoskeletal components.

DETECTION OF DENSE INTRA- AND PERINUCLEAR 10 NM FILAMENT SYSTEMS BY WHOLE MOUNT AND EMBEDMENT-FREE ELECTRON MICROSCOPY IN SEVERAL SPECIES OF THE GREEN ALGAL ORDER DASYCLADALES

SummaryIn contrast to the immense body of evidence supporting the occurrence of intermediate filament (IF) proteins in the animal kingdom, there is only limited information on their distribution in plants. Nevertheless, a number of immunocytochemical and electron microscopical observations indicate that particularly in higher plant cells IFs contribute to the construction of the cyto- and karyoskeleton. Here we show by whole mount electron microscopy of the giant nuclei extruded together with adhering cytoplasm from the rhizoids of some species of the algal order Dasycladales that cytoplasmic 10 nm filament networks also occur in unicellular, mononucleated green organisms of early evolutionary origin. The filament systems were associated with the residual nuclear envelope which consisted of a dense arrangement of pore complexes suspended by a meshwork of short 5 to 6 nm filaments; structurally it was very similar to the nuclear envelopes obtained from mammalian cells. When the Dasycladales nuclei were processed side by side with mouse skin fibroblasts, the algal filament systems were physically almost indistinguishable from the mammalian vimentin filament network. Embedment-free thin sections of rhizoids have not only confirmed the existence of the perinculear 10 nm filaments and their seamless association with the nuclear envelope, but have demonstrated the existence of an extensive intranuclear meshwork of 10 nm filaments. The latter were morphologically indistinguishable from the perinuclear 10 nm filaments and seem to be connected to these via the nuclear envelope to form a continuum. Among a variety of antibodies directed against mammalian IF proteins, only polyclonal anti-mouse lamin B antibodies decorated the cytoplasmic filaments of the Dasycladales cells. Surprisingly, none of the antibodies decorated the thinner filaments of the nuclear envelope, which possibly represent the nuclear lamina. In accord with this observation, one anti-lamin B antibody recognized in Western blot analysis of a urea extract ofAcetabularia acetabulum rhizoids three polypeptides with Mrs of approximately 47,000, 64,000, and 76,000. The proteins did not react with the α-IFA antibody. Since the Dasycladales have a fossil record of nearly 600 million years — an extant genus, Acicularia, also investigated here, evolved about 170 million years ago -, the molecular characterization of the subunit proteins of their cytoplasmic filament systems might throw further light on the evolution and biological role of IFs.

Pepstatin A: Polymerization of an oligopeptide

Pepstatin A, a pentapeptide with the molecular weight of 686, is a naturally occurring inhibitor of aspartyl proteases secreted by Streptomyces species. Above a critical concentration of 0.1 mM at low ionic strength and neutral pH, it can polymerize into filaments which may extend over several micrometers. After negative staining, these filaments show a helical substructure with characteristic diameters ranging from 6 to 12 nm. Selected images at higher magnification suggest the filaments are composed of two intertwined 6 nm strands. This is in agreement with the optical diffraction analysis which additionally established a periodic pitch of 25 nm for the helical intertwining. Rotary shadowing of the pepstatin A filaments clearly demonstrated the right-handedness of the helical twist. In physiological salt solution or at higher concentrations of pepstatin A, a variety of higher order structures were observed, including ribbons, sheets and cylinders with both regular and twisted or irregular geometries. Pepstatin A can interact with intermediate filament subunit proteins. These proteins possess a long, alpha-helical rod domain that forms coiled-coil dimers, which through both hydrophobic and ionic interactions form tetramers which, in turn, in the presence of physiological salt concentrations, polymerize into the 10 nm intermediate filaments. In the absence of salt, pepstatin A and intermediate filament proteins polymerize into long filaments with a rough surface and a diameter of 15-17 nm. This polymerization appears to be primarily driven by nonionic interactions between pepstatin A and polymerization-competent forms of intermediate filament proteins, resulting in a composite filament. Polymerization-incompetent proteolytic fragments of vimentin, lacking portions of the head and/or tail domain, failed to copolymerize with pepstatin A into long filaments under these conditions. These peptides, as well as bovine serum albumin, were found to stick to the surface of pepstatin A filaments, ribbons and sheets. Independent evidence for direct association of pepstatin A with intermediate filament subunit proteins was provided not only by electron microscopy but also by UV difference spectra. Pepstatin A loses its ability to inhibit the aspartyl protease of the human immunodeficiency virus type 1 following polymerization into the higher order structures described here. The amazing fact that pepstatin A can spontaneously self-associate to form very large polymers seems to be a more rare event for such small peptides. The other examples of synthetic or naturally occurring oligopeptides discussed in this review which are able to polymerize into higher order structures possess a common property, their hydrophobicity, often manifested by clusters of valine or isoleucine residues.(ABSTRACT TRUNCATED AT 400 WORDS)