John F. Hess

A Juvenile-Onset, Progressive Cataract Locus on Chromosome 3q21-q22 Is Associated with a Missense Mutation in the Beaded Filament Structural Protein–2

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.

Autosomal-dominant congenital cataract associated with a deletion mutation in the human beaded filament protein gene BFSP2

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.

cDNA analysis of the 49 kDa lens fiber cell cytoskeletal protein: a new, lens-specific member of the intermediate filament family?

Two proteins, with molecular weights of 49 (CP49) and 115 kDa (CP115) as judged by SDS PAGE, have been shown by immunocytochemistry to be components of the beaded filament, a cytoskeletal structure thus far demonstrated only in the lens fiber cell. We have used antibodies reactive with CP49 to screen a mouse lens cDNA expression library. An immunoreactive clone with an approximately 1.0 kb insert was identified and purified. DNA sequence analysis shows the presence of an open reading frame that extends from the upstream cloning site for 660 bases. Contained within this reading frame are 2 peptide sequences nearly identical to 2 peptide sequences obtained from purified bovine CP49. Northern analysis revealed that the mRNA for the CP49 is not detectable in mouse brain, muscle, lung, liver, or heart. A search of the Genbank database showed that the partial cDNA sequence for the murine CP49 is unique, but that this partial sequence shows a strong similarity to multiple members of the intermediate filament family, with greatest similarity to type I acidic cytokeratins. The data presented here suggests that the CP49 is related to, and possibly represents a new member of the intermediate filament family. These data, in concert with previously published work, suggests 1) the CP49 and CP115, which have been localized to the beaded filament, are related to the family of IF proteins, and 2) these two proteins comprise a cytoskeletal structure which is structurally distinct from classical 8-11 nm intermediate filaments, thus possibly comprising a structurally novel form of intermediate filament.

Real-time Observation of Coiled-coil Domains and Subunit Assembly in Intermediate Filaments

We have utilized electron paramagnetic resonance spectroscopy to study secondary structure, subunit interaction, and molecular orientation of vimentin molecules within intact intermediate filaments and assembly intermediates. Spectroscopy data prove α-helical coiled-coil structures at individual amino acids 316–336 located in rod 2B. Analysis of positions 305, 309, and 312 identify this region as conforming to the helical pattern identified within 316–336 and thus demonstrates that, contrary to some previous predictions, this region is in an α-helical conformation. We show that by varying the position of the spin label, we can identify both intra- and inter-dimer interactions. With a label attached to the outside of the α-helix, we have been able to measure interactions between positions 348 of separate dimers as they align together in intact filaments, identifying the exact point of overlap. By mixing different spin-labeled proteins, we demonstrate that the interaction at position 348 is the result of an anti-parallel arrangement of dimers. This approach provides high resolution structural information (<2 nm resolution), can be used to identify molecular arrangements between subunits in an intact intermediate filament, and should be applicable to other noncrystallizable filamentous systems as well as to the study of protein fibrils.

The structure of vimentin linker 1 and rod 1B domains characterized by site-directed spin-labeling electron paramagnetic resonance (SDSL-EPR) and X-ray crystallography.

Background: The complete structure is not known for any intermediate filament (IF) protein. Results: Linker 1 and rod 1B in human vimentin were characterized using electron paramagnetic resonance spectroscopy and x-ray crystallography. Conclusion: The rod 1B adopts two functional conformations that mediate formation of an anti-parallel “A11” tetramer. Significance: Understanding vimentin structure provides insight into all IFs and the related human pathologies. Despite the passage of ∼30 years since the complete primary sequence of the intermediate filament (IF) protein vimentin was reported, the structure remains unknown for both an individual protomer and the assembled filament. In this report, we present data describing the structure of vimentin linker 1 (L1) and rod 1B. Electron paramagnetic resonance spectra collected from samples bearing site-directed spin labels demonstrate that L1 is not a flexible segment between coiled-coils (CCs) but instead forms a rigid, tightly packed structure. An x-ray crystal structure of a construct containing L1 and rod 1B shows that it forms a tetramer comprising two equivalent parallel CC dimers that interact with one another in the form of a symmetrical anti-parallel dimer. Remarkably, the parallel CC dimers are themselves asymmetrical, which enables them to tetramerize rather than undergoing higher order oligomerization. This functionally vital asymmetry in the CC structure, encoded in the primary sequence of rod 1B, provides a striking example of evolutionary exploitation of the structural plasticity of proteins. EPR and crystallographic data consistently suggest that a very short region within L1 represents a minor local distortion in what is likely to be a continuous CC from the end of rod 1A through the entirety of rod 1B. The concordance of this structural model with previously published cross-linking and spectral data supports the conclusion that the crystallographic oligomer represents a native biological structure.

Site-directed Spin Labeling and Electron Paramagnetic Resonance Determination of Vimentin Head Domain Structure

Intermediate filament (IF) proteins have been predicted to have a conserved tripartite domain structure consisting of a largely α-helical central rod domain, flanked by head and tail domains. However, crystal structures have not been reported for any IF or IF protein. Although progress has been made in determining central rod domain structure, no structural data have been reported for either the head or tail domains. We used site-directed spin labeling and electron paramagnetic resonance to analyze 45 different spin labeled mutants spanning the head domain of vimentin. The data, combined with results from a previous study, provide strong evidence that the polypeptide backbones of the head domains form a symmetric dimer of closely apposed backbones that fold back onto the rod domain, imparting an asymmetry to the dimer. By following the behavior of spin labels during the process of in vitro assembly, we show that head domain structure is dynamic, changing as a result of filament assembly. Finally, because the vimentin head domain is the major site of the phosphorylation that induces disassembly at mitosis, we studied the effects of phosphorylation on head domain structure and demonstrate that phosphorylation drives specific head domain regions apart. These data provide the first evidence-based model of IF head domain structure.

Intermediate Filaments Regulate Tissue Size and Stiffness in the Murine Lens

PURPOSE To define the contributions of the beaded filament (BF), a lens-specific intermediate filament (IF), to lens morphology and biomechanics. METHODS Wild-type and congenic CP49 knockout (KO) mice were compared by using electrophysiological, biomechanical, and morphometric approaches, to determine changes that occurred because of the absence of this cytoskeletal structure. RESULTS Electrophysiological assessment established that the fiber cells lacking the lens-specific IFs were indistinguishable from wild-type fiber cells. The CP49 KO mice exhibited lower stiffness, and an unexpected higher resilience than the wild-type lenses. The absence of these filaments resulted in lenses that were smaller, and exhibited a higher ratio of lens:lens nucleus size. Finally, lens shape differed as well, with the CP49 KO showing a higher ratio of axial:equatorial diameter. CONCLUSIONS Previous work has shown that BFs are necessary in maintaining fiber cell and lens structural phenotypes with age, and that absence of these filaments results in a loss of lens clarity. This work demonstrates that several tissue-level properties that are critical to lens function are also dependent, at least in part, on the presence of these lens-specific IFs.

Electron paramagnetic resonance analysis of the vimentin tail domain reveals points of order in a largely disordered region and conformational adaptation upon filament assembly

Very little data have been reported that describe the structure of the tail domain of any cytoplasmic intermediate filament (IF) protein. We report here the results of studies using site directed spin labeling and electron paramagnetic resonance (SDSL‐EPR) to explore the structure and dynamics of the tail domain of human vimentin in tetramers (protofilaments) and filaments. The data demonstrate that in contrast to the vimentin head and rod domains, the tail domains are not closely apposed in protofilaments. However, upon assembly into intact IFs, several sites, including positions 445, 446, 451, and 452, the conserved “beta‐site,” become closely apposed, indicating dynamic changes in tail domain structure that accompany filament elongation. No evidence is seen for coiled‐coil structure within the region studied, in either protofilaments or assembled filaments. EPR analysis also establishes that more than half of the tail domain is very flexible in both the assembly intermediate and the intact IF. However, by positioning the spin label at distinct sites, EPR is able to identify both the rod proximal region and sites flanking the beta‐site motif as rigid locations within the tail. The rod proximal region is well assembled at the tetramer stage with only slight changes occurring during filament elongation. In contrast, at the beta site, the polypeptide backbone transitions from flexible in the assembly intermediate to much more rigid in the intact IF. These data support a model in which the distal tail domain structure undergoes significant conformational change during filament elongation and final assembly.

Head and rod 1 interactions in vimentin: identification of contact sites, structure, and changes with phosphorylation using site-directed spin labeling and electron paramagnetic resonance.

We have used site-directed spin labeling (SDSL) and electron paramagnetic resonance (EPR) to identify residues 17 and 137 as sites of interaction between the head domain and rod domain 1A of the intermediate filament protein vimentin. This interaction was maximal when compared with the spin labels placed at up- and downstream positions in both head and rod regions, indicating that residues 17 and 137 were the closest point of interaction in this region. SDSL EPR characterization of residues 120-145, which includes the site of head contact with rod 1A, reveals that this region exhibits the heptad repeat pattern indicative of α-helical coiled-coil structure, but that this heptad repeat pattern begins to decay near residue 139, suggesting a transition out of coiled-coil structure. By monitoring the spectra of spin labels placed at the 17 and 137 residues during in vitro assembly, we show that 17-137 interaction occurs early in the assembly process. We also explored the effect of phosphorylation on the 17-137 interaction and found that phosphorylation-induced changes affected the head-head interaction (17-17) in the dimer, without significantly influencing the rod-rod (137-137) and head-rod (17-137) interactions in the dimer. These data provide the first direct evidence for, and location of, headrod interactions in assembled intermediate filaments, as well as direct evidence of coiled-coil structure in rod 1A. Finally, the data identify changes in the structure in this region following in vitro phosphorylation.

Sequence features accurately predict genome-wide MeCP2 binding in vivo

Methyl-CpG binding protein 2 (MeCP2) is critical for proper brain development and expressed at near-histone levels in neurons, but the mechanism of its genomic localization remains poorly understood. Using high-resolution MeCP2-binding data, we show that DNA sequence features alone can predict binding with 88% accuracy. Integrating MeCP2 binding and DNA methylation in a probabilistic graphical model, we demonstrate that previously reported genome-wide association with methylation is in part due to MeCP2s affinity to GC-rich chromatin, a result replicated using published data. Furthermore, MeCP2 co-localizes with nucleosomes. Finally, MeCP2 binding downstream of promoters correlates with increased expression in Mecp2-deficient neurons.