FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2102272153> ?p ?o ?g. }

• W2102272153 endingPage "5108" @default.
• W2102272153 startingPage "5101" @default.
• W2102272153 abstract "Previous studies have shown that rodent neurofilaments (NF) are obligate heteropolymers requiring NF-L plus either NF-M or NF-H for filament formation. We have assessed the competence of human NF-L and NF-M to assemble and find that unlike rat NF-L, human NF-L is capable of self-assembly. However, human NF-M cannot form homopolymers and requires the presence of NF-L for incorporation into filaments. To investigate the stage at which filament formation is blocked, the rod domains or the full-length subunits of human NF-L, human NF-M, and rodent NF-L were analyzed in the yeast “interaction trap” system. These studies demonstrated that the fundamental block to filament formation in those neurofilaments that do not form homopolymers is at the level of dimer formation. Based on theoretical biophysical considerations of the requirements for the formation of coiled-coil structures, we predicted which amino acid differences were likely to be responsible for the differing dimerization potentials of the rat and human NF-L rod domains. We tested these predictions using site-specific mutagenesis. Interestingly, single amino acid changes in the rod domains designed to restore or eliminate the coiled-coil propensity were found respectively to convert rat NF-L into a subunit capable of homopolymerization and human NF-L into a protein that is no longer able to self-assemble. Our results additionally suggest that the functional properties of the L12 linker region of human NF-L, generally thought to assume an extended β-sheet conformation, are consonant with an α-helix that positions the heptad repeats before and after it in an orientation that allows coiled-coil dimerization. These studies reveal an important difference between the assembly properties of the human and rodent NF-L subunits possibly suggesting that the initiating events in neurofilament assembly may differ in the two species. Previous studies have shown that rodent neurofilaments (NF) are obligate heteropolymers requiring NF-L plus either NF-M or NF-H for filament formation. We have assessed the competence of human NF-L and NF-M to assemble and find that unlike rat NF-L, human NF-L is capable of self-assembly. However, human NF-M cannot form homopolymers and requires the presence of NF-L for incorporation into filaments. To investigate the stage at which filament formation is blocked, the rod domains or the full-length subunits of human NF-L, human NF-M, and rodent NF-L were analyzed in the yeast “interaction trap” system. These studies demonstrated that the fundamental block to filament formation in those neurofilaments that do not form homopolymers is at the level of dimer formation. Based on theoretical biophysical considerations of the requirements for the formation of coiled-coil structures, we predicted which amino acid differences were likely to be responsible for the differing dimerization potentials of the rat and human NF-L rod domains. We tested these predictions using site-specific mutagenesis. Interestingly, single amino acid changes in the rod domains designed to restore or eliminate the coiled-coil propensity were found respectively to convert rat NF-L into a subunit capable of homopolymerization and human NF-L into a protein that is no longer able to self-assemble. Our results additionally suggest that the functional properties of the L12 linker region of human NF-L, generally thought to assume an extended β-sheet conformation, are consonant with an α-helix that positions the heptad repeats before and after it in an orientation that allows coiled-coil dimerization. These studies reveal an important difference between the assembly properties of the human and rodent NF-L subunits possibly suggesting that the initiating events in neurofilament assembly may differ in the two species. Intermediate filaments (IFs) 1The abbreviations used are: IFs, intermediate filaments; aa, amino acid(s); NF, neurofilament(s); PBS, phosphate-buffered saline. are a heterogeneous family of proteins sharing common structural features that can be subdivided into six types (I–VI) based on sequence homology (1Steinert P.M. Roop D.R. Annu. Rev. Biochem. 1986; 57: 593-625Google Scholar). IF genes are expressed in a cell type-specific and developmentally regulated manner with cells frequently containing only a single IF type at a particular stage of differentiation. Neurofilaments (NFs) are the predominant IF in mature neurons but are preceded during neuronal differentiation by a succession of other IFs including vimentin (2Cochard P. Paulin D. J. Neurosci. 1984; 4: 2080-2094Google Scholar) nestin (49Lendahl U. Zimmerman L.B. McKay R.D.G. Cell. 1990; 60: 585-595Google Scholar), α-internexin (3Kaplan M.P. Chin S.S.M. Fliegner K.H. Liem R.K.H. J. Neurosci. 1990; 10: 2735-2748Google Scholar, 4Fliegner K.H. Kaplan M.P. Wood T.L. Pintar J.E. Liem R.K. J. Comp. Neurol. 1994; 342: 161-173Google Scholar), and peripherin (5Parysek L.M. Chisholm R.L. Ley C.A. Goldman R.D. Neuron. 1988; 1: 395-401Google Scholar). Neurofilaments are assemblies of three subunits, the NF-L (molecular mass, 68 kDa), NF-M (150 kDa), and NF-H (200 kDa) (6Eagles P.A.M. Pant H.C. Gainer H. Goldman R.D. Steinert P.M. Cellular and Molecular Biology of Intermediate Filaments. Plenum Publishing Corp., New York1990: 37-94Google Scholar). These three components form heteropolymeric 10-nm filaments that run parallel along the length of the axon with frequent cross-bridges between neighboring filaments. Axonal neurofilaments are thought to serve a primarily structural function. Evidence from a Japanese quail (quiverer) with a spontaneous mutation in NF-L (7Ohara O. Gahara Y. Miyake T. Teraoka H. Kitamura T. J. Cell Biol. 1993; 121: 387-395Google Scholar) and a line of transgenic mice expressing an NF-H-β galactosidase fusion protein (8Eyer J. Peterson A. Neuron. 1994; 12: 389-405Google Scholar) suggest that a loss of axonal neurofilaments results in a decreased axonal diameter. The first step in filament formation is the lateral associations of the α-helical rod domains via hydrophobic interactions to form a coiled-coil dimer (9Fuchs E. Weber K. Annu. Rev. Biochem. 1994; 63: 345-382Google Scholar). The rod consists of an α-helix that is interrupted by three short non-helical linker sequences (L1, L12, and L2). Heptad repeats of hydrophobic amino acids confer an amphipathic character to the α-helical domain that allows coiled-coil interactions between compatible IF molecules. This may result in homodimer formation in the case of vimentin or obligate heterodimer formation in the case of type I and type II keratins (9Fuchs E. Weber K. Annu. Rev. Biochem. 1994; 63: 345-382Google Scholar). Both in vitro and in vivo studies have probed the ability of individual neurofilament proteins to form homo- and heteropolymers. Purified bovine (10Balin B.J. Lee V.M.-Y. Brain Res. 1991; 556: 196-208Google Scholar, 11Hisanaga S. Hirokawa N. J. Mol. Biol. 1988; 202: 297-305Google Scholar, 12Liem R. Hutchison S. Biochemistry. 1982; 21: 3216-3221Google Scholar, 13Lifsics M.R. Williams R.C. Biochemistry. 1984; 23: 2866-2875Google Scholar, 14Gardner E.E. Dahl D. Bignami A. J. Neurosci. Res. 1984; 11: 145-155Google Scholar, 15Moon H.M. Wisniewski T. Merz P. De Martini J. Wisniewski H.M. J. Cell Biol. 1981; 89: 560-567Google Scholar, 16Zackroff R.V. Idler W.W. Steinert P.M. Goldman R.D. Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 754-757Google Scholar), porcine (17Geisler N. Weber K. J. Mol. Biol. 1981; 151: 565-571Google Scholar, 18Minami Y. Endo S. Sakai H. J. Biochem. (Tokyo). 1984; 96: 1481-1490Google Scholar), and murine NF-L (19Heins S. Wong P.C. Muller S. Goldie K. Cleveland D.W. Aebi U. J. Cell Biol. 1993; 123: 1517-1533Google Scholar) and, to a lesser degree, NF-M and NF-H (10Balin B.J. Lee V.M.-Y. Brain Res. 1991; 556: 196-208Google Scholar, 14Gardner E.E. Dahl D. Bignami A. J. Neurosci. Res. 1984; 11: 145-155Google Scholar) assemblein vitro into 10-nm homopolymers (20Balin B.J. Clark E.A. Trojanowski J.Q. Lee V.M.-Y. Brain Res. 1991; 556: 181-195Google Scholar). In contrast, rodent neurofilament proteins expressed individually in cells that lack an endogenous intermediate filament network (SW13 vim− cells) are unable to form homopolymers, yet can form 10-nm filaments when coexpressed with NF-M or NF-H (21Ching G. Liem R. J. Cell Biol. 1993; 122: 1323-1335Google Scholar, 22Lee M. Xu Z. Wong P. Cleveland D. J. Cell Biol. 1993; 122: 1337-1350Google Scholar) or when expressed in cells containing an endogenous vimentin network, through assembly with vimentin (21Ching G. Liem R. J. Cell Biol. 1993; 122: 1323-1335Google Scholar, 23Chin S.M. Liem R.K.H. Eur. J. Cell Biol. 1989; 50: 475-490Google Scholar, 24Monteiro M.J. Cleveland D.W. J. Cell Biol. 1989; 108: 579-593Google Scholar). In the present work we examine the human NF-L and NF-M and derivatives of them in SW13 (vim−) cells and in the yeast “interaction trap” system. Our results demonstrate that human NF-L is capable of self-assembly, an important distinction from the rodent NF-L subunits which are obligate heteropolymers. Human NF-M or truncations of it that contained the rod domain could not polymerize in the absence of NF-L. Our analysis shows that two distinct structural features of the human NF-L rod are important determinants of its ability to homodimerize. First, human NF-L has more charged residues next to the hydrophobic residues of the heptad repeats in its rod domain. These charged residues are believed to stabilize coiled-coil interaction by electrostatic attraction between the strands. Second, the combination of the absence of a serine residue (compared with the rodent NF-L) and the presence of a nearby proline residue allows the L12 linker region of human NF-L to mimic an α-helix with a heptad repeat. The same region of rat NF-L contains the additional serine, and its conformation is predicted to be an extended β-structure (25Parry D.A.D. Goldman R.D. Steinert P.M. Cellular and Molecular Biology of Intermediate Filaments. Plenum Publishing Corp., New York1990: 175-204Google Scholar). Since dimer formation is likely to be the initiating event in filament formation, our results suggest that this important intracellular event may be different in human than in rodents. The plasmid pNF-L contains a complete human NF-L gene plus 2.8 kilobase pairs of upstream sequence. The plasmid pRSV-NF-L containing a full-length rat NF-L cDNA driven by a Rous sarcoma virus promoter was obtained from Dr. R. Liem (21Ching G. Liem R. J. Cell Biol. 1993; 122: 1323-1335Google Scholar). A plasmid containing a complete human NF-M genomic clone (pNF-M) has been previously described (26Lee V.M.-Y. Elder G.A. Chen L.-C. Liang Z. Snyder S.E. Friedrich V.L. Lazzarini R.A. Mol. Brain Res. 1992; 15: 76-84Google Scholar). To create a “tagged” human NF-M, a nucleotide sequence encoding an 11 amino acid epitope tag (Ala-Ser-Met-Thr-Gly-Gly-Gln-Gln-Met-Gly-Arg) was inserted at either amino acid 21 (amino-terminal tagged, designated pSS019) or amino acid 444 (internal tagged, designated pSS028) of the plasmid pNF-M (numbering according to Ref. 50Myers M. Lazzarini R.A. Lee V.M.-Y. Schlaepfer W.W. Nelson D.L. EMBO J. 1987; 6: 1617-1626Google Scholar). The internal tagged NF-M plasmids were used to prepare the following series of deletions: Δamino-terminal domain (amino acids 22–80 deleted); Δglutamic acid-rich domain (aa 451–611 deleted); Δglutamic acid and multiphosphorylation repeat (aa 551–790 deleted); and Δcarboxyl-terminal domain (aa 690–914 deleted). AnNF-M-L-M hybrid gene was constructed by removing amino acids 89–444 from pNF-M and replacing it with a polymerase chain reaction-amplified NF-L rod domain (aa 83–414). The rod region of the final product was verified by sequencing. For yeast interaction trap experiments, fragments encoding NF rod domains (human NF-L amino acids 85–410, human NF-M amino acids 98–421, and rat NF-L amino acids 86–415) or full-length proteins were cloned into the vectors pGBT9 and pGAD42, (27Fields S. Ok-kyu S. Nature. 1989; 340: 245-246Google Scholar) using oligonucleotides to preserve open reading frames. Amino acids were inserted, deleted, or substituted using the Quikchange site-directed mutagenesis kit (Stratagene). Changes were verified by sequencing on an Applied Biosystems automated DNA sequencer. Human SW13 (vim−) cells were obtained from Dr. R. Evans and cultured as described (28Sarria A. Nordeen S. Evans R. J. Cell Biol. 1990; 111: 553-565Google Scholar). Cells were transiently transfected by electroporation (440 V, 500 microfarads) with 20 μg of DNA per 10-cm dish based on the protocol of van den Hoff et al. (29van den Hoff M.J.B. Moorman A.F. Lamers W.H. Nucleic Acids Res. 1992; 20: 2902Google Scholar). Following electroporation, cells were plated directly into Lab-Tek 4-well glass chamber slides (Nunc) and incubated for 48 h before immunostaining. Cells grown on glass chamber slides were washed three times in phosphate-buffered saline (PBS) and fixed with 2% paraformaldehyde in PBS for 15 min at room temperature. The paraformaldehyde was removed with three rinses of PBS, and nonspecific binding was blocked by treatment for 1 h with PGBA (PBS, 0.1% gelatin, 1% bovine serum albumin), 2% goat serum, and 0.1% saponin. Treated slides were incubated for 60 and 30 min, respectively, with primary and secondary antibodies diluted in PGBA, 2% goat serum, and 0.1% saponin. Unbound primary and secondary antibodies were removed with three 10-min washes in PBS at room temperature. Nuclei were counterstained by inclusion of 4′,6-diamidino-2-phenylindole hydrochloride with the secondary antibody. Slides were mounted in 90% glycerol + diazabicyclo-octane and analyzed using laser scanning confocal microscopy (Leica TCS confocal microscope). A rabbit polyclonal anti-NF-L (Dr. V. Lee, University of Pennsylvania), mouse monoclonal anti-TAG (ascites), and a rabbit anti-vimentin (Sigma) all diluted 1:100 were used as primary antibodies. Donkey anti-mouse Texas Red-conjugated (Amersham Corp.) and donkey anti-rabbit fluorescein-conjugated (Amersham Corp.) secondary antibodies were used at a 1:200 dilution. The yeast vectors pGAD424 or pGBT9 containing cDNAs encoding the rod domains or full-length forms of human NF-L, human NF-M, human NF-M/L/M, and rat NF-L were introduced into the yeast strain Y526 (30Becker D.M. Guarente L. Methods Enzymol. 1991; 194: 182-187Google Scholar) by electroporation. Cells were permeabilized and β-galactosidase activity assayed according to the protocol of Guarente (48Guarente L. Methods Enzymol. 1983; 101: 181-191Google Scholar). Sequences of vimentins, desmins, keratins, and neurofilament light chains were retrieved from the GenBankTM data base of nucleotide sequences using STRINGSEARCH in the GCG package (31Genetics Computer Group Wisconsin Package, Version 9.0. Genetics Computer Group, Inc., Madison, WI1996Google Scholar), and complete genomic sequences and mRNA sequences were translated to protein sequences. The resulting sequences of 6 keratins, 4 desmins, 12 vimentins, and 3 NF-Ls, including human and rat, were aligned using the PILEUP module of the GCG package (31Genetics Computer Group Wisconsin Package, Version 9.0. Genetics Computer Group, Inc., Madison, WI1996Google Scholar). The sequence alignment was inspected to identify heptad repeats defining coiled-coils (32Ballesteros J.A. Weinstein H. Methods Neurosci. 1995; 25: 366-428Google Scholar) and differences between rat and human NF-L; a total of eight differences was identified in the rod sequence. Models of the coiled-coil portion of human and rat NF-L were constructed using the crystal structure of the GCN4 leucine zipper (33O'Shea E.K. Klemm J.D. Kim P.S. Alber T. Science. 1991; 254: 539-544Google Scholar) as a template. The GCN4 sequence was mutated to the selected pieces of human and rat NF-L containing differences considered most likely to be responsible for disrupting dimerization. The residues were modeled in allowed rotameric conformations, and the structures were searched manually for allowed rotamers that avoid steric clashes in the model structure. This low resolution model was used to explore possible proximities of residues and to identify the nature of interactions in the rat NF-L sequence that may destabilize the dimerization process. The propensities of specific amino acids to occupy particular positions in the heptad repeat (34Cohen C. Parry D.A.D. Proteins Struct. Funct. Genet. 1990; 7: 1-15Google Scholar) and the structural role of Pro residues in helices (32Ballesteros J.A. Weinstein H. Methods Neurosci. 1995; 25: 366-428Google Scholar, 35Sankararamakrishnan R. Vishveshwara S. Biopolymers. 1990; 30: 287-298Google Scholar, 36Sankararamakrishnan R. Vishveshwara S. Int. J. Peptide Protein Res. 1992; 39: 356-363Google Scholar) were used as criteria for selecting likely candidates for disruptive interactions. For the helical part of the structure, the backbone and dihedral angles were obtained as average dihedral angle values from the crystal structure of the GCN4 leucine zipper, with values of −63.0 and −42.5, respectively. The Pro-kink segment between position a in heptad 2 to positiond in heptad 5 (Fig. 6) were modeled with the parameters defined in Table I. The resulting structure of this distorted α-helix in that region was close to a 310 helix turn.Table IBackbone dihedral angle values used in the model structure of the Pro-kink region in the L12 subunitPositionDihedral angleφψωi − 8−64.8−9.8180i − 7−65.2−9.9180i − 6−65.2−10.1180i − 5−65.2−9.9180i − 4−63.0−8.3180i − 3−48.3−15.5−172.4i − 2−75.9−54.6−174.3i − 1−54.6−32.2171.7Pro (i)−57.2−43.9180 Open table in a new tab The ability of human NF-L to self-assemble into filaments was assessed in human SW13 (vim−) cells that lack an endogenous intermediate filament network (28Sarria A. Nordeen S. Evans R. J. Cell Biol. 1990; 111: 553-565Google Scholar). These cells were transfected with a plasmid (pNF-L) containing the entire humanNF-L gene plus 2.8 kilobase pairs of upstream sequence. Under our conditions we obtain an average transfection efficiency of 15–20%. NF-L and vimentin expression was monitored by immunofluorescence 48 h following transfection. Transfection of human NF-L alone resulted in extensive filament formation throughout the cytoplasm in the absence of any other IF protein (Fig. 1 A). Staining with anti-vimentin antibodies revealed that 1–4% of the cells were vimentin positive. Others (22Lee M. Xu Z. Wong P. Cleveland D. J. Cell Biol. 1993; 122: 1337-1350Google Scholar) have reported comparable levels of spontaneous reversion in this cell line. However, double labeling with antibodies to NF-L and vimentin demonstrated that filament formation by human NF-L was not dependent on the presence of vimentin (data not shown). These results contrast sharply with those reported previously for rodent NF subunits which showed that the rat and mouse NF-L proteins do not assemble into filaments under similar conditions (21Ching G. Liem R. J. Cell Biol. 1993; 122: 1323-1335Google Scholar,22Lee M. Xu Z. Wong P. Cleveland D. J. Cell Biol. 1993; 122: 1337-1350Google Scholar). This difference between our results and those of others cannot be attributed to systematic or technical differences in the experiments. We have transfected SW13 (vim−) cells with the ratnf-l gene used by Ching and Liem (21Ching G. Liem R. J. Cell Biol. 1993; 122: 1323-1335Google Scholar) and obtained results that are in complete agreement with theirs: the rat NF-L protein distributed uniformly throughout the cytoplasm and no filamentous structures present (Fig. 1 B). Thus, differences in the ability of human and rodent NF-L to homopolymerize appear to reflect potentially important and fundamental differences that are traceable to the few differences in the amino acid sequences of the two proteins. We carried out similar transfection experiments using human NF-M to determine if the ability to homopolymerize was present in other human NF subunits or was restricted to NF-L. The plasmid pSS028 contains a modified humanNF-M gene that encodes an NF-M protein containing an 11-amino acid tag sequence (Ala-Ser-Met-Thr-Gly-Gly-Gln-Gln-Met-Gly-Arg) inserted at amino acid 444 (internal tag). The inclusion of this epitope tag allowed the simultaneous detection of both phosphorylated and non-phosphorylated forms of the protein with an anti-tag monoclonal antibody. Our previous studies have demonstrated that the tag does not interfere with expression or polymerization of NF-M in transgenic mice or transfected cells and that filaments containing the tag appear to function normally (37Elder G.A. Friedrich V.L. Liang Z. Li X. Lazzarini R.A. Mol. Brain Res. 1994; 26: 177-188Google Scholar). Expression of the tagged human NF-M protein in SW13 (vim−) cells did not result in the formation of filamentous networks. Immunofluorescence staining using anti-tag antibody revealed that the human NF-M protein was uniformly distributed throughout the cytoplasm in a diffuse and at times granular pattern (Fig. 1 C). In this regard, the human NF-M behaves similarly to the rodent NF-L and NF-M subunits. Interestingly, rat NF-L and human NF-M can participate in filament formation through heteropolymerization. Double transfection experiments were performed in which human NF-M was coexpressed with either human or rat NF-L. Cells transfected with human NF-L and human NF-M contained an extensive filamentous network throughout the cytoplasm that contained both NF subunits co-localized within filaments (Fig. 2, A and C). In Fig. 2 C, the finer filaments corresponding to those labeled with antibodies directed at the human NF-L do not appear in the photomicrograph because the gain of the photomultiplier was set so that the main fibers reveal good detail. At higher gain setting the full array of fine filaments was visible. Expression of rat NF-L with human NF-M also resulted in extensive filament formation and co-localization of both proteins (Fig. 2, B and D) indicating the assembly competence of each subunit, but only as a heteropolymer. Inhibitory regions in parts of the molecule not directly involved in homophilic interactions could explain the incompetence of human NF-M to homopolymerize. To address this possibility, NF-M genes encoding truncated forms of the tagged human protein were prepared and transfected into SW13 (vim−) cells. The following series of deletions were examined: Δamino-terminal domain (amino acids 22–80 deleted); Δglutamic acid-rich domain (aa 451–611); Δglutamic acid-rich and multiphosphorylation repeat (aa 551–790); Δcarboxyl-terminal domain (aa 690–914). Expression of these truncated proteins was assessed using anti-tag monoclonal antibodies and, where appropriate, the anti-NF antibodies SMI-31 and SMI-32 which detect phosphorylation-dependent and independent epitopes of the human NF-M, respectively (38Lee V.M.-Y. Otvos Jr., L. Carden M. Hollosi M. Dietzschold B. Lazzarini R.A. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 1998-2002Google Scholar). In all cases staining was diffuse and distributed throughout the cytoplasm without evidence of filament formation (data not shown). However, none of the deletions affected the ability of human NF-M to form heteropolymers since filaments formed when the deletions were coexpressed with human NF-L (data not shown). The failure of human NF-M to form homopolymeric filaments might indicate the incompetence of its rod domain to form dimers. If so, then replacement of the NF-M rod with the human NF-L rod should allow homopolymerization of the hybrid molecule. To test this hypothesis a hybrid NF-M-L-M gene was created which encodes a protein with the amino- and carboxyl-terminal domains of NF-M but the rod domain of human NF-L. When this construct was expressed alone in SW13 (vim−) cells, a diffuse granular staining was apparent throughout the cytoplasm, and no filament assembly was detected. However, when the NF-M-L-M protein was coexpressed with human NF-L, extensive filament formation occurred. These results indicate that the amino- and carboxyl-terminal sequences of NF-M might interfere with homopolymerization, but the failure of the native NF-M to homopolymerize cannot be solely attributed to this interference since the NF-M rod domain without flanking sequences does not exhibit significant homophilic interactions in the yeast interaction trap analyses. The immunocytochemical analyses described above show that human NF-L, but not NF-M, is capable of homopolymeric assembly into filaments of a dimension and complexity that can be visualized by light microscopy. Filament assembly is a multistep process, and the block to NF-M homofilament assembly might occur at steps after the initial interactions between neurofilament subunits. We investigated the earliest steps in NF subunit interactions using the yeast “two-hybrid” or interaction trap system, similar to that used by Meng et al. (39Meng J. Khan S. Ip W. J. Biol. Chem. 1996; 271: 1599-1604Google Scholar) to investigate homodimer formation by vimentin. In this system, expression of a reporter gene (Escherichia coli lacZ gene) is controlled by a transactivator complex consisting of two hybrid proteins. A functional transactivating complex is achieved when a DNA binding domain, supplied by one hybrid subunit, and an activation domain, supplied by the second, are held in close proximity by the adhesive domains of two hybrid proteins. We examined the interactions between various NF subunits or their rod domains by incorporating them as the adhesive components in the two-hybrid complex. cDNA sequences encoding human NF-L, human NF-M, rat NF-L, or only the α-helical rod domains of each were separately fused to the activation or DNA binding subunits of the transactivating complex. All pairwise combinations of activation subunits with DNA binding subunits were tested for interactions by double transfections into yeast. The results of these experiments are summarized in Table II, where strongly positive results are presented in bold type. The results shown in the first two vertical columns indicate that full-length human NF-M or its rod domain interact strongly with only full-length human NF-L or its rod domain. All combinations of human NF-M (rod domain or whole) with itself or rat NF-L (rod domain or whole) failed to interact. These results suggest that the yeast two-hybrid assay is a more stringent test of interaction than the SW13 transfection assay, since the human NF-M was able to form filaments with the rat NF-L in the latter assay (Fig. 2, B and D). It is possible that the yeast two-hybrid system places steric constraints on the association that are not necessary for filament formation. The third and fourth vertical columns show that full-length human NF-L or its rod interact very strongly with the whole subunits or the rod domains of human NF-L, human NF-M, or rat NF-L. The last two columns indicate that rat NF-L or its rod domain interact with human NF-L or its rod domain while failing to interact with itself or human NF-M or its rod domain. The results shown in Table II were obtained with permeabilized yeast cells and have been normalized for differences in culture turbidity. It should be noted that each NF domain was tested as a fusion partner in both the activation and DNA binding subunits, and all pairwise combinations were tested for transactivation. The results of these tests do not show any significant bias between reciprocal pairs of hybrid proteins. We conclude from these analyses that human NF-L but not rodent NF-L or human NF-M is capable of significant homodimer formation.Table IIInteraction-trap analysis of various neurofilament subunits and their rod domainsT-hMT-hMrodT-hLT-hLrodT-rLrodT-rLD-hM0.00.081.291.680.040.0D-hMrod0.00.141.591.950.00.08D-hL0.80.924.293.041.321.37D-hLrod0.570.412.843.151.261.32D-rL0.00.022.551.580.040.09D-rLrod0.00.082.141.640.040.05 Open table in a new tab Based on theoretical biophysical considerations, we attempted to determine which of the amino acid differences between human and rat NF-L could be responsible for the differences in their homopolymerization ability. Fig. 3 shows alignments of the human and rat NF-L rod domains and indicates the position of the α-helical (1A, 1B, 2A, and 2B) and linker (L1, L2, and L12) regions (25Parry D.A.D. Goldman R.D. Steinert P.M. Cellular and Molecular Biology of Intermediate Filaments. Plenum Publishing Corp., New York1990: 175-204Google Scholar). The human and rat NF-L rod sequences are highly homologous with only 8 amino acid differences (indicated by a * in Fig. 3). Two regions in particular were noted which could account for the difference in homopolymerization. The first was at aa 161 in the human protein which is an arginine and the corresponding aa position 162 in the rat which is a glutamine. This difference occurs at position e of the heptad, next to a hydrophobic surface created by the heptad repeat, where it is likely to affect the corresponding electrostatic attractions between two rod domains forming a coiled-coil. Such charged residues (usually Arg, Lys, or Glu) are considered to be responsible for additional attractive interactions between helices forming a coiled-coil (34Cohen C. Parry D.A.D. Proteins Struct. Funct. Genet. 1990; 7: 1-15Google Scholar) and are also major contributors to the characteristic stagger for a given protein. To test this hypothesis an arginine was substituted for the glutamine at position 162 in the rat rod. The reciprocal substitution of Arg161 for Gln161 was carried out in the human NF-L rod. When transfected into SW13 (vim−) cells, the human NF-L (Gln161) did not form filaments (Fig. 4 A), whereas the rat NF-L (Arg162) formed filamen" @default.
• W2102272153 created "2016-06-24" @default.
• W2102272153 creator A5001337906 @default.
• W2102272153 creator A5001870291 @default.
• W2102272153 creator A5014370221 @default.
• W2102272153 creator A5016775860 @default.
• W2102272153 creator A5032840137 @default.
• W2102272153 creator A5084958891 @default.
• W2102272153 date "1998-02-01" @default.
• W2102272153 modified "2023-10-16" @default.
• W2102272153 title "Neurofilament (NF) Assembly; Divergent Characteristics of Human and Rodent NF-L Subunits" @default.
• W2102272153 cites W1473407224 @default.
• W2102272153 cites W1512160880 @default.
• W2102272153 cites W1554920990 @default.
• W2102272153 cites W1566478858 @default.
• W2102272153 cites W1593180325 @default.
• W2102272153 cites W169938888 @default.
• W2102272153 cites W1814239271 @default.
• W2102272153 cites W1833104430 @default.
• W2102272153 cites W1967024359 @default.
• W2102272153 cites W1967365672 @default.
• W2102272153 cites W1977186059 @default.
• W2102272153 cites W1980581336 @default.
• W2102272153 cites W1981271792 @default.
• W2102272153 cites W1982922864 @default.
• W2102272153 cites W1983048518 @default.
• W2102272153 cites W1986578258 @default.
• W2102272153 cites W1987145582 @default.
• W2102272153 cites W1995467090 @default.
• W2102272153 cites W1995674610 @default.
• W2102272153 cites W1998238515 @default.
• W2102272153 cites W2005193885 @default.
• W2102272153 cites W2007434688 @default.
• W2102272153 cites W2009145359 @default.
• W2102272153 cites W2009696832 @default.
• W2102272153 cites W2014802721 @default.
• W2102272153 cites W2016870828 @default.
• W2102272153 cites W2017581486 @default.
• W2102272153 cites W2023246522 @default.
• W2102272153 cites W2032861753 @default.
• W2102272153 cites W2054861715 @default.
• W2102272153 cites W2055290772 @default.
• W2102272153 cites W2059035007 @default.
• W2102272153 cites W2061027966 @default.
• W2102272153 cites W2083856144 @default.
• W2102272153 cites W2087687043 @default.
• W2102272153 cites W2090095487 @default.
• W2102272153 cites W2091488232 @default.
• W2102272153 cites W2094775681 @default.
• W2102272153 cites W2098330936 @default.
• W2102272153 cites W2119297613 @default.
• W2102272153 cites W2122412289 @default.
• W2102272153 cites W2125594344 @default.
• W2102272153 cites W2143499422 @default.
• W2102272153 cites W2145229125 @default.
• W2102272153 cites W2159470622 @default.
• W2102272153 cites W2171602378 @default.
• W2102272153 doi "https://doi.org/10.1074/jbc.273.9.5101" @default.
• W2102272153 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/9478962" @default.
• W2102272153 hasPublicationYear "1998" @default.
• W2102272153 type Work @default.
• W2102272153 sameAs 2102272153 @default.
• W2102272153 citedByCount "69" @default.
• W2102272153 countsByYear W21022721532012 @default.
• W2102272153 countsByYear W21022721532013 @default.
• W2102272153 countsByYear W21022721532014 @default.
• W2102272153 countsByYear W21022721532015 @default.
• W2102272153 countsByYear W21022721532016 @default.
• W2102272153 countsByYear W21022721532017 @default.
• W2102272153 countsByYear W21022721532019 @default.
• W2102272153 countsByYear W21022721532020 @default.
• W2102272153 countsByYear W21022721532021 @default.
• W2102272153 countsByYear W21022721532022 @default.
• W2102272153 countsByYear W21022721532023 @default.
• W2102272153 crossrefType "journal-article" @default.
• W2102272153 hasAuthorship W2102272153A5001337906 @default.
• W2102272153 hasAuthorship W2102272153A5001870291 @default.
• W2102272153 hasAuthorship W2102272153A5014370221 @default.
• W2102272153 hasAuthorship W2102272153A5016775860 @default.
• W2102272153 hasAuthorship W2102272153A5032840137 @default.
• W2102272153 hasAuthorship W2102272153A5084958891 @default.
• W2102272153 hasBestOaLocation W21022721531 @default.
• W2102272153 hasConcept C104292427 @default.
• W2102272153 hasConcept C104317684 @default.
• W2102272153 hasConcept C157207234 @default.
• W2102272153 hasConcept C185592680 @default.
• W2102272153 hasConcept C18903297 @default.
• W2102272153 hasConcept C203014093 @default.
• W2102272153 hasConcept C204232928 @default.
• W2102272153 hasConcept C2777730290 @default.
• W2102272153 hasConcept C2778914748 @default.
• W2102272153 hasConcept C55493867 @default.
• W2102272153 hasConcept C62478195 @default.
• W2102272153 hasConcept C86803240 @default.
• W2102272153 hasConcept C95444343 @default.
• W2102272153 hasConceptScore W2102272153C104292427 @default.
• W2102272153 hasConceptScore W2102272153C104317684 @default.
• W2102272153 hasConceptScore W2102272153C157207234 @default.

• next