The epidermal intermediate filament proteins of tunicates are distant keratins; a polymerisation-competent hetero coiled coil of the Styela D protein and Xenopus keratin 8.

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.

FTIR-Spectroscopy of multistranded coiled coil proteins.

Coiled coils of different order were investigated using infrared (IR) spectroscopy. Recently, we demonstrated that dimeric coiled coils display unique vibrational spectra with at least three separable bands instead of only one band of a classical alpha-helix in the amide I region. This was attributed to a distortion of the helical structure by the supercoil bending, giving rise to bands that are not observed in the undistorted helix. Here, we investigated coiled coils forming trimers, tetramers, and pentamers. These higher order coiled coils, in general, possess larger superhelical pitches, resulting in a smaller helical distortion. We found that all coiled coils studied, including the native dimeric GCN4 leucine zipper and its variants leading to parallel trimers and tetramers as well as the rod portions of fibritin (parallel trimer), alpha-actinin (antiparallel spectrin type trimer), and COMP (parallel pentamer), displayed the typical three band pattern of the coiled coil amide I spectra. However, the separation of these three bands and their positional deviation from the classical alpha-helical band position was correlated to the extent of the helical distortion as reflected by the pitch values of the supercoils. The most pronounced spectral anomaly was found for the tropomyosin dimer with a reported helical pitch of 137 A, whereas the smallest spectral distortion was found for the pentameric COMP complex and the tetrameric leucine zipper mutant, both with a pitch of about 205 A.

Assembly and architecture of invertebrate cytoplasmic intermediate filaments reconcile features of vertebrate cytoplasmic and nuclear lamin-type intermediate filaments.

The two major intermediate filament (IF) proteins from the esophagus epithelium of the snail Helix pomatia and the two major IF proteins from muscle tissue of the nematode Ascaris suum were investigated under a variety of assembly conditions. The lowest-order complexes from each of the four protostomic invertebrate (p-INV) IF proteins are parallel, unstaggered dimers involving two-stranded alpha-helical coiled coil formation of their approximately 350 amino acid residue central rod domain (i.e. long-rod). In the electron microscope these are readily recognized by their distinct approximately 56 nm long rod with two globular domains (i.e. representing the non-helical carboxy-terminal tail domain of the p-INV IF proteins) attached at one end, closely resembling vertebrate lamin dimers. The next-higher-order oligomers are tetramers, which are easily recognized by their two pairs of globular tail domains attached at either end of a approximately 72 nm long central rod portion. According to their size and shape, these tetramers are built from two dimers associated laterally in an antiparallel, approximately half-staggered fashion via the amino-terminal halves of their rod domains. This is similar to the NN-type tetramers found as the most abundant oligomer species in all types of vertebrate cytoplasmic IF proteins, which contain a approximately 310 amino acid residue central rod domain (i.e. short-rod). As a first step toward filament formation, the p-INV IF tetramers anneal longitudinally into protofilaments by antiparallel CC-type association of the carboxy-terminal halves of their dimer rods. The next step involves radial growth, occurring initially through lateral association of two four-chain protofilaments into octameric subfibrils, which then further associate into mature, full-width filaments. Head-to-tail polymers of dimers and paracrystalline fibers commonly observed with vertebrate lamins were only rarely seen with p-INV IF proteins. The globular domains residing at the carboxy-terminal end of p-INV IF dimers were studding the surface of the filaments at regular, approximately 24.5 nm intervals, thereby giving them a "beaded" appearance with an axial periodicity of about 24.5 nm, which is approximately 3 nm longer than the corresponding approximately 21.5 nm repeat pattern exhibited by short-rod vertebrate IFs.

Peptides from the conserved ends of the rod domain of desmin disassemble intermediate filaments and reveal unexpected structural features: a circular dichroism, Fourier transform infrared, and electron microscopic study.

Synthetic peptides representing the conserved ends of the rod domain of desmin are shown to disassemble preformed desmin filaments when added in moderate molar excess. This argues for a similar importance of both ends of the rod for filament stability. Recent structural models of intermediate filaments suggest close proximity of the ends and perhaps even an interaction (N. Geisler, J. Schünemann, and K. Weber, 1992, Eur. J. Biochem. 206, 841-852; P. M. Steinert, L. N. Marekov, R. D. B. Fraser, and D. A. D. Parry, 1993, J. Mol. Biol. 230, 436-452). Since the disassembling activity of the peptides, in addition to their sequences, should be related in some way to their secondary structure, we have investigated the structures of a number of related peptides which all arise from the ends of the rod using electron microscopic and spectroscopic methods. All peptides showed the expected alpha-helical structure at low concentrations in the presence of trifluoroethanol, as revealed by circular dichroism. At higher concentrations the peptides showed extensive self-aggregation into various types of filaments. The filaments contain the peptides in beta-sheet conformation as shown by Fourier transform infrared spectroscopy.

Chemical cross-linking indicates a staggered and antiparallel protofilament of desmin intermediate filaments and characterizes one higher-level complex between protofilaments.

Tetrameric rods, protofilaments and assembled filaments of desmin, the intermediate filament protein of muscle, have been chemically cross-linked with the lysine specific cross-linkers EGS [ethylene glycol bis(succinimidylsuccinate), 1.61 nm span] and bis(sulfosuccinimidyl) suberate (1.14 nm span). One bis(sulfosuccinimidyl)suberate and two EGS cross-links were isolated from the rod and characterized. They show that the two coiled coils in the rod tetramer are staggered by approximately 15-20 nm and strongly indicate an antiparallel arrangement in which the inner overlapping part of the rod is formed by the amino-terminal helices 1A, 1B and 2A. Both EGS cross-links identified in the rod were also isolated from cross-linked filaments. The isolated rod, therefore, represents a complex also present in identical, or very similar form in protofilaments and in assembled filaments. Cross-linked filaments yielded a third EGS cross-link that must have been formed between neighboring protofilaments. It connects the highly conserved carboxy-terminus of helix 2B of the first protofilament to the overlap region formed by helices 1A and 2A of the second protofilament. The restrictions posed by these cross-links on current filament models are discussed.

[Synthesis and CNS- activity of cis-pyrano[2,3-b]-[1,4]dioxane derivatives ]. / Synthese und ZNS-Wirkungen von cis-Pyrano[2,3-b]-[1,4]-dioxanderivaten.

The alcohols 2, the amines 5, 7 and 10, the indole- and quinoline-derivatives 13 and 14, the enamines 3 and 4 and the silylenolethers 17 and 18 were prepared starting with the title compound 1. The 1-(7-phenyl-7-pyranodioxinyl)-piperidines 10-cis and 10-trans show striking CNS-activity. The transisomer is about twice as active as the cis-isomer.

[Intramolecular aromatic alkylation. 28. Synthesis and pharmacological testing of homologized and hydroxylated 3,4-dihydro-1'-methylspiro[naphthalin-1(2H),4'-piperidine]. / Synthese und pharmakologische Prüfung homologer und hydroxylierter 3,4-Dihydro-1'-methylspiro[naphthalin-1(2H),4'-piperidine].

The compounds 4, 8, and 10, prepared by standard methods, are converted to the phenylpyridylakanes 9; their methoiodides 11 are reduced to yield the tetrahydropyridines 12. Whereas the cyclisation of 12a gives the isomers 1f and 1g, 12b furnishes only the isomer 1i or 1k, respectively. H3PO4 is found to be more suitable than HBr to cyclize 12c and 12d to the homologues 1l and 1m. Treatment of 12c and 12d affords the bromides 13a and 13b by HBr-addition, which are isolated for the first time. Compounds 1l, 1b, 1c, and 1a exhibit a notable activity in the writhing test, whereas the hydroxylated derivatives 1h, 1e, and 1f are definitely less active. The homologue 1m is inactive.

[Intramolecular aromatic alkylation. 24. Synthesis and pharmacologic testing of N-alkyldecahydro-7H-naphthioisoquinolines]. / Intramolekulare Aromatenalkylierungen, 24.Mitt.: Synthese und pharmakologische Prüfung von N-Alkyl-decahydro-7H-naphthoisochinolinen.

Demethylation of 1a,b is achieved by trichloroethyl chloroformate via the carbamates 1d,f to yield the secondary bases 1e,g which are realkylated directly by selected alkyl halogenides (route A) or via the acid amides 1k,m by LiAlH4-reduction (route B) to give 1h,i,o and 1l,n respectively. Pharmacological examination indicates substance 1a to be a powerful molecule in the writhing test, whereas substitution of the aromatic moiety and exchange of the N-methyl group in 1b,c,i diminish the activity distinctly.