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Feather keratin has a composite structure with a filament-matrix texture, and transmission electron microscopy studies of thin transverse sections of feather rachis by Rogers and Filshie in the early 1960s showed that the filaments have a strong tendency to form sheets. Potentially this could account for the unusual X-ray diffraction pattern noted by Bear and Rugo in the early 1950s, which was interpreted by them as indicating a two-dimensional net structure. Although it is 50years since these major advances were made the possibility of extracting information on the nature of the filament packing from the diffraction pattern has never been explored. The present contribution shows how, when taken together with current information on the nature of β-sheets in feather keratin, certain features of the X-ray diffraction pattern can now be used to determine the likely arrangement of the filaments in the sheet., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Infantile myopathies with diaphragmatic paralysis are genetically heterogeneous, and clinical symptoms do not assist in differentiating between them. We used phased haplotype analysis with subsequent targeted exome sequencing to identify MEGF10 mutations in a previously unidentified type of infantile myopathy with diaphragmatic weakness, areflexia, respiratory distress and dysphagia. MEGF10 is highly expressed in activated satellite cells and regulates their proliferation as well as their differentiation and fusion into multinucleated myofibers, which are greatly reduced in muscle from individuals with early onset myopathy, areflexia, respiratory distress and dysphagia.

Familial biparental hydatidiform mole (FBHM) is the only known pure maternal-effect recessive inherited disorder in humans. Affected women, although developmentally normal themselves, suffer repeated pregnancy loss because of the development of the conceptus into a complete hydatidiform mole in which extraembryonic trophoblastic tissue develops but the embryo itself suffers early demise. This developmental phenotype results from a genome-wide failure to correctly specify or maintain a maternal epigenotype at imprinted loci. Most cases of FBHM result from mutations of NLRP7, but genetic heterogeneity has been demonstrated. Here, we report biallelic mutations of C6orf221 in three families with FBHM. The previously described biological properties of their respective gene families suggest that NLRP7 and C6orf221 may interact as components of an oocyte complex that is directly or indirectly required for determination of epigenetic status on the oocyte genome., (Copyright © 2011 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

Anterior segment dysgenesis describes a group of heterogeneous developmental disorders that affect the anterior chamber of the eye and are associated with an increased risk of glaucoma. Here, we report homozygous mutations in peroxidasin (PXDN) in two consanguineous Pakistani families with congenital cataract-microcornea with mild to moderate corneal opacity and in a consanguineous Cambodian family with developmental glaucoma and severe corneal opacification. These results highlight the diverse ocular phenotypes caused by PXDN mutations, which are likely due to differences in genetic background and environmental factors. Peroxidasin is an extracellular matrix-associated protein with peroxidase catalytic activity, and we confirmed localization of the protein to the cornea and lens epithelial layers. Our findings imply that peroxidasin is essential for normal development of the anterior chamber of the eye, where it may have a structural role in supporting cornea and lens architecture as well as an enzymatic role as an antioxidant enzyme in protecting the lens, trabecular meshwork, and cornea against oxidative damage., (Copyright © 2011 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

Numerous inherited diseases develop due to missense mutations, leading to an amino-acid substitution. Whether an amino-acid change is pathogenic depends on the level of deleterious effects caused by the amino-acid alteration. We show an example of different structural and phenotypic consequences caused by two individual amino-acid changes at the same position. Epidermolysis bullosa simplex (EBS) is a genodermatosis resulting from KRT5 or KRT14 mutations. Mutation analysis of an EBS family revealed that affected individuals were heterozygous for a, to our knowledge, previously unreported mutation of c.1237G>C (p.Ala413Pro) in KRT14. Interestingly, 2 of 100 unrelated normal controls were heterozygous, and 1 of the 100 was homozygous for a different mutation in this position, c.1237G>A (p.Ala413Thr). In silico modeling of the protein demonstrated deleterious structural effects from proline substitution but not from threonine substitution. In vitro transfection studies revealed a significantly larger number of keratin-clumped cells in HaCaT cells transfected with mutant KRT14 complementary DNA (cDNA) harboring p.Ala413Pro than those transfected with wild-type KRT14 cDNA or mutant KRT14 cDNA harboring p.Ala413Thr. These results show that changes in two distinct amino acids at a locus are destined to elicit different phenotypes due to the degree of structural distortion resulting from the amino-acid alterations.

Purpose: To investigate whether three consanguineous families from the Punjab province of Pakistan, with affected members with recessively inherited congenital cataract microcornea with corneal opacity, are genetically homogeneous., Methods: An ophthalmic examination was performed on each family member to establish the diagnosis. The two largest families were analyzed by homozygosity mapping using SNP arrays. Linkage was confirmed using polymorphic microsatellite markers, and logarithm of odds (LOD) scores were calculated. Candidate genes were prioritized using the ENDEAVOUR program., Results: Autosomal recessive congenital cataract-microcornea with corneal opacity mapped to chromosome 10cen for family MEP57 and to either chromosomes 2ptel or 20p for family MEP60. For MEP57, the refined interval was 36.8 Mb flanked by D10S1208 (35.3 Mb) and D10S676 (72.1 Mb). For MEP60, the interval containing the mutation was either 6.7 Mb from the telomere of chromosome 2 to marker D2S281 or 3.8 Mb flanked by D20S906 (1.5 Mb) and D20S835 (5.3 Mb). Maximum multipoint LOD scores of 3.09, 1.94, and 3.09 were calculated at D10S567, D2S281, and D20S473 for families MEP57 and MEP60. Linkage to these loci was excluded for family MEP68. SLC4A11 was excluded as a candidate gene for the observed phenotype in MEP60., Conclusions: The authors have identified two new loci, one on chromosome 10cen and the other on 2ptel or 20p, that are associated with recessively inherited congenital cataract-microcornea with corneal opacity. This phenotype is genetically heterogeneous in the Pakistani population. Further genetic studies of this kind, combined with a detailed phenotypic description, will contribute to more precise classification criteria for anterior segment disease.

Eukaryotic cilia and flagella are vital sensory and motile organelles. The calcium channel PKD2 mediates sensory perception on cilia and flagella, and defects in this can contribute to ciliopathic diseases. Signaling from Pkd2-dependent Ca²+ rise in the cilium to downstream effectors may require intermediary proteins that are largely unknown. To identify these proteins, we carried out genetic screens for mutations affecting Drosophila melanogaster sperm storage, a process mediated by Drosophila Pkd2. Here we show that a new mutation lost boys (lobo) encodes a conserved flagellar protein CG34110, which corresponds to vertebrate Ccdc135 (E = 6e-78) highly expressed in ciliated respiratory epithelia and sperm, and to FAP50 (E = 1e-28) in the Chlamydomonas reinhardtii flagellar proteome. CG34110 localizes along the fly sperm flagellum. FAP50 is tightly associated with the outer doublet microtubules of the axoneme and appears not to be a component of the central pair, radial spokes, dynein arms, or structures defined by the mbo waveform mutants. Phenotypic analyses indicate that both Pkd2 and lobo specifically affect sperm movement into the female storage receptacle. We hypothesize that the CG34110/Ccdc135/FAP50 family of conserved flagellar proteins functions within the axoneme to mediate Pkd2-dependent processes in the sperm flagellum and other motile cilia.

Avian hard keratin has a filament-matrix texture in which the filaments contain a helical array of twisted β-sheets and the matrix has unusually high concentrations of cysteine, glycine, and tyrosine. X-ray diffraction studies have established that similar filaments exist in the hard keratins of crocodiles, turtles, tuataras, lizards and snakes. Here, the relationship between amino acid sequence and the filament-matrix texture is explored in a wide variety of avian and reptilian hard keratins. Universally, the molecules contain three distinct domains: a central domain rich in β-favoring residues associated with the filament framework, and N- and C-terminal domains associated with the matrix and with crosslinking via disulfide bonds. A variety of structural probes were employed to identify the β-framework of the filaments and a common pattern 34 residues in length was found in all cases. In addition, detailed analyses of the sequences in the two "matrix" domains revealed profound differences between the Archosaurs (birds, crocodiles and turtles), where the N-terminal domains were very similar, and the Squamates (snakes and lizards) where the N-terminal domains varied widely in length and composition, in some cases exhibiting a subdomain structure, and segments of highly homologous sequence. The C-terminal domains in both branches varied widely in composition but almost all exhibit a subdomain structure characterized by a terminal sequence rich in cysteine and arginine residues. A revised model for the molecular organization in avian and reptilian hard keratins is presented and similarities and differences in the matrix domains are noted., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Background: Mutations in WDR72 have been identified in autosomal recessive hypomaturation amelogenesis imperfecta (AI)., Objective: to describe a novel WDR72 mutation and report the ultrastructural enamel phenotype associated with a different WDR72 mutation., Methods: A family segregating autosomal recessive hypomaturation AI was recruited, genomic DNA obtained and WDR72 sequenced. Four deciduous teeth from one individual with a previously published WDR72 mutation, extracted as part of clinical care, were subjected to scanning electron microscopy, energy-dispersive X-ray analysis and transverse microradiography., Results: A novel homozygous nonsense mutation, R897X, was identified in WDR72 in a family originating from Pakistan. Ultrastructural analysis of enamel from the deciduous teeth of an AI patient with the WDR72 mutation S783X revealed energy-dispersive X-ray analysis spectra with normal carbon and nitrogen peaks, excluding retention of enamel matrix protein. However, transverse microradiography values were significantly lower for affected teeth when compared to normal teeth, consistent with reduced mineralisation. On scanning electron microscopy the enamel rod form observed was normal, yet with inter-rod enamel more prominent than in controls. This appearance was unaltered following incubation with either α-chymotrypsin or lipase., Conclusions: The novel WDR72 mutation described brings the total reported WDR72 mutations to four. Analyses of deciduous tooth enamel in an individual with a homozygous WDR72 mutation identified changes consistent with a late failure of enamel maturation without retention of matrix proteins. The mechanisms by which intracellular WDR72 influences enamel maturation remain unknown., (Copyright © 2010 S. Karger AG, Basel.)

The rod domain of IF molecules has been characterized as four alpha-helical coiled-coil segments (1A, 1B, 2A and 2B), three linkers (L1, L12 and L2) and a stutter at the centre of segment 2B. Two of these breaks in coiled-coil continuity (L2 and stutter) have been modelled on the basis of structural data obtained from related proteins. Subsequently, X-ray crystallographic studies on fragments of IF molecules have shown that both models were correct. The third of the breaks - L1 - was predicted to have a flexible structure, consistent with observations that the head domain can fold back over segments 1A and 1B and also unwind into separate strands. Here the structure of the fourth discontinuity (L12) has been modelled. For most IF chain types two conformations are proposed for an eight-residue motif that displays a quasi two-residue repeat based on the presence of apolar residues. In IF it is proposed that the motif will adopt an alpha-helical conformation but that in the molecule the conformation will be beta-like. Thus, assembly will result in or result from a conformational change in L12 thereby attributing L12 a more dynamic and important role in assembly than expected., (Copyright 2009 Elsevier Inc. All rights reserved.)

We have investigated the expression of 52 of the 54 keratins in beard hair medulla. We found that not only 12 hair keratins but, unexpectedly, also 12 epithelial keratins are potentially expressed in medulla cells. The latter comprise keratins also present in outer- and inner-root sheaths and in the companion layer. Keratins K5, K14, K17, K25, K27, K28, and K75 define a "pre-medulla," composed of cells apposed to the upper dermal papilla. Besides K6, K16, K7, K19, and K80, all pre-medullary epithelial keratins continue to be expressed in the medulla proper, along with the 12 hair keratins. Besides this unique feature of cellular keratin co-expression, the keratin pattern itself is highly variable in individual medulla cells. Remarkably, both epithelial and hair keratins behave highly promiscuously with regard to heterodimer- and IF formation, which also includes keratin chain interactions in IF bundles. We also identified cortex cells within the medullary column. These exhibit all the properties of genuine cortex cells, including a particular type of keratin heterogeneity of their compact IF bundles. In both keratin expression profile and keratin number, medulla cells are distinct from all other cells of the hair follicle or from any other epithelium.

Background: Nonsense mutations in FAM83H are a recently described underlying cause of autosomal dominant (AD) hypocalcified amelogenesis imperfecta (AI)., Objective: This study aims to report a novel c.1374C>A p.Y458X nonsense mutation and describe the associated ultrastructural phenotype of deciduous teeth., Methods: A family of European origin from the Iberian Peninsula with AD-inherited AI was ascertained. Family members were assessed through clinical examination and supporting investigations. Naturally exfoliated deciduous teeth from 2 siblings were investigated by scanning electron microscopy (SEM), energy dispersive X-ray analysis (EDX) and transverse microradiography (TMR)., Results: On clinical and radiographic investigation the appearances of the affected deciduous and permanent teeth were consistent with hypocalcified AI with small focal areas of more normal looking enamel. DNA sequencing identified a novel c.1374C>A p.Y458X FAM83H nonsense mutation in affected, but not in either unaffected family members or unrelated controls. Exfoliated teeth were characterised by substantial post-eruptive enamel loss on gross examination. Irregular, poor quality enamel prisms were observed on SEM. These were coated in amorphous material. TMR and EDX confirmed reduced mineral and increased organic content in enamel, respectively., Conclusions: FAM83H nonsense mutations have recently been recognised as a cause of AD hypocalcified AI. We report a novel nonsense FAM83H mutation and describe the associated preliminary ultrastructural phenotype in deciduous teeth. This is characterised by poorly formed enamel rods with inappropriate retention of amorphous material, which is likely to represent retained organic matrix that contributes to the overall hypomineralised phenotype., (2009 S. Karger AG, Basel.)

Healthy dental enamel is the hardest and most highly mineralized human tissue. Though acellular, nonvital, and without capacity for turnover or repair, it can nevertheless last a lifetime. Amelogenesis imperfecta (AI) is a collective term for failure of normal enamel development, covering diverse clinical phenotypes that typically show Mendelian inheritance patterns. One subset, known as hypomaturation AI, is characterised by near-normal volumes of organic enamel matrix but with weak, creamy-brown opaque enamel that fails prematurely after tooth eruption. Mutations in genes critical to enamel matrix formation have been documented, but current understanding of other key events in enamel biomineralization is limited. We investigated autosomal-recessive hypomaturation AI in a consanguineous Pakistani family. A whole-genome SNP autozygosity screen identified a locus on chromosome 15q21.3. Sequencing candidate genes revealed a point mutation in the poorly characterized WDR72 gene. Screening of WDR72 in a panel of nine additional hypomaturation AI families revealed the same mutation in a second, apparently unrelated, Pakistani family and two further nonsense mutations in Omani families. Immunohistochemistry confirmed intracellular localization in maturation-stage ameloblasts. WDR72 function is unknown, but as a putative beta propeller is expected to be a scaffold for protein-protein interactions. The nearest homolog, WDR7, is involved in vesicle mobilization and Ca2+-dependent exocytosis at synapses. Vesicle trafficking is important in maturation-stage ameloblasts with respect to secretion into immature enamel and removal of cleaved enamel matrix proteins via endocytosis. This raises the intriguing possibility that WDR72 is critical to ameloblast vesicle turnover during enamel maturation.

Primary congenital glaucoma (PCG) is an autosomal-recessive condition characterized by high intraocular pressure (IOP), usually within the first year of life, which potentially could lead to optic nerve damage, globe enlargement, and permanent loss of vision. To date, PCG has been linked to three loci: 2p21 (GLC3A), for which the responsible gene is CYP1B1, and 1p36 (GLC3B) and 14q24 (GLC3C), for which the genes remain to be identified. Here we report that null mutations in LTBP2 cause PCG in four consanguineous families from Pakistan and in patients of Gypsy ethnicity. LTBP2 maps to chromosome 14q24.3 but is around 1.3 Mb proximal to the documented GLC3C locus. Therefore, it remains to be determined whether LTBP2 is the GLC3C gene or whether a second adjacent gene is also implicated in PCG. LTBP2 is the largest member of the latent transforming growth factor (TGF)-beta binding protein family, which are extracellular matrix proteins with multidomain structure. It has homology to fibrillins and may have roles in cell adhesion and as a structural component of microfibrils. We confirmed localization of LTBP2 in the anterior segment of the eye, at the ciliary body, and particularly the ciliary process. These findings reveal that LTBP2 is essential for normal development of the anterior chamber of the eye, where it may have a structural role in maintaining ciliary muscle tone.

Cone-rod dystrophy (CRD) is an inherited progressive retinal dystrophy affecting the function of cone and rod photoreceptors. By autozygosity mapping, we identified null mutations in the ADAM metallopeptidase domain 9 (ADAM9) gene in four consanguineous families with recessively inherited early-onset CRD. We also found reduced photoreceptor responses in Adam9 knockout mice, previously reported to be asymptomatic. In 12-month-old knockout mice, photoreceptors appear normal, but the apical processes of the retinal pigment epithelium (RPE) cells are disorganized and contact between photoreceptor outer segments (POSs) and the RPE apical surface is compromised. In 20-month-old mice, there is clear evidence of progressive retinal degeneration with disorganized POS and thinning of the outer nuclear layer (ONL) in addition to the anomaly at the POS-RPE junction. RPE basal deposits and macrophages were also apparent in older mice. These findings therefore not only identify ADAM9 as a CRD gene but also identify a form of pathology wherein retinal disease first manifests at the POS-RPE junction.

Syncoilin is a 64kDa intermediate filament (IF) protein expressed in myocytes at the sarcolemma, perinucleus, myotendenous and neuromuscular junctions. Here we present a revised domain projection and structural analysis for the original isoform (sync-1) and introduce two novel syncoilin isoforms (sync-2 and sync-3) generated by exon splicing. On the basis of consensus identity we propose that syncoilin be reclassified as a type III IF protein. All three syncoilin isoforms lack a L1 domain, a significant departure from standard IF rod domain projections that is likely to impact significantly on their biological function. Our analyses indicate that syncoilin is unlikely to form classical intermediate filament structures by itself, and that the significant difference in C-terminal structure between the three isoforms indicates that they may play divergent roles in myocytes. We show that despite lacking an apparent structural role in striated muscle, syncoilin isoforms are differentially and strongly upregulated in response to cardiotoxin induced regeneration and denervation induced atrophy in the C57BL/6 mouse, possibly suggesting an atypical role for syncoilin in muscle.

The combination of recessively inherited cone-rod dystrophy (CRD) and amelogenesis imperfecta (AI) was first reported by Jalili and Smith in 1988 in a family subsequently linked to a locus on chromosome 2q11, and it has since been reported in a second small family. We have identified five further ethnically diverse families cosegregating CRD and AI. Phenotypic characterization of teeth and visual function in the published and new families reveals a consistent syndrome in all seven families, and all link or are consistent with linkage to 2q11, confirming the existence of a genetically homogenous condition that we now propose to call Jalili syndrome. Using a positional-candidate approach, we have identified mutations in the CNNM4 gene, encoding a putative metal transporter, accounting for the condition in all seven families. Nine mutations are described in all, three missense, three terminations, two large deletions, and a single base insertion. We confirmed expression of Cnnm4 in the neural retina and in ameloblasts in the developing tooth, suggesting a hitherto unknown connection between tooth biomineralization and retinal function. The identification of CNNM4 as the causative gene for Jalili syndrome, characterized by syndromic CRD with AI, has the potential to provide new insights into the roles of metal transport in visual function and biomineralization.

alpha-Helical coiled coils are remarkable for the diversity of related conformations that they adopt in both fibrous and globular proteins, and for the range of functions that they exhibit. The coiled coils are based on a heptad (7-residue), hendecad (11-residue) or a related quasi-repeat of apolar residues in the sequences of the alpha-helical regions involved. Most of these, however, display one or more sequence discontinuities known as stutters or stammers. The resulting coiled coils vary in length, in the number of chains participating, in the relative polarity of the contributing alpha-helical regions (parallel or antiparallel), and in the pitch length and handedness of the supercoil (left- or right-handed). Functionally, the concept that a coiled coil can act only as a static rod is no longer valid, and the range of roles that these structures have now been shown to exhibit has expanded rapidly in recent years. An important development has been the recognition that the delightful simplicity that exists between sequence and structure, and between structure and function, allows coiled coils with specialized features to be designed de novo.

Objective: To evaluate the incidence and type of intracranial and inner ear abnormalities in children with sensorineural hearing loss (SNHL) identified with magnetic resonance imaging (MRI) and stratified by the degree and type of SNHL., Design: Retrospective review of medical records and MRIs., Setting: Tertiary care children's hospital., Patients: A total of 227 children aged 1 month to 17 years (mean age, 5.3 years; male to female ratio, 1:1) with a diagnosis of SNHL underwent MRI from June 1,1996, to June 1, 2002. Of these children, 170 had clinical information available and technically adequate MRIs and were included in the study., Intervention: Magnetic resonance imaging., Main Outcome Measure: Identification of an abnormality of the intracranial contents, inner ear, and cochlear nerve., Results: Of the 170 children, 101 (59%) had bilateral SNHL and 69 (41%) had unilateral SNHL, comprising 271 ears with SNHL. Abnormalities of the inner ear were found in 108 ears (40%) with 87 (32%) having abnormalities of the cochlea, which were considered mild in 63 (23%) and moderate to severe in 24 (9%). Forty-nine of 271 ears (18%) with SNHL demonstrated an either absent (26/49 [53%]) or deficient (23/49 [47%]) cochlear nerve. Ears with severe and profound SNHL had more abnormalities than ears with mild and moderate SNHL (66/138 [48%] vs 23/80 [29%]; P = .006), and children having ears with unilateral moderate, severe, or profound SNHL had more inner ear abnormalities than children with bilateral moderate, severe, or profound SNHL (28/45 [62%] vs 54/144 [38%]; P = .004)., Conclusions: The overall incidence of inner ear abnormalities in ears of children with SNHL evaluated by MRI is 40%. The most common abnormalities seen were an abnormal cochlea and abnormal cochlear nerve. Children with severe and profound SNHL have a greater percentage of inner ear anomalies than children with mild or moderate SNHL. Children with unilateral hearing loss have a greater percentage of inner ear anomalies than children with bilateral SNHL.

Avian feathers have a filament-matrix texture and X-ray diffraction studies show that the filament has a helical structure with four repeating units per turn. Each repeating unit consists of a pair of twisted beta-sheets related by a perpendicular diad, and the twist in the sheets is of opposite hand to that of the helix. Each sheet is believed to comprise a 32-residue segment of the feather keratin molecule, which contains around 100 residues, the remainder constituting the matrix. In the present contribution, the sequence of emu feather is mapped to the low-resolution model derived earlier from X-ray studies. This shows that the inner surface of the "beta-sandwich" is densely populated by hydrophobic residues and that the charged residues and cysteine residues lie on the outer surface. In addition, the inner residues in the repeating unit mesh neatly together in layers oriented perpendicular to the filament axis. Amino acid sequences from a range of avian and reptilian keratins were collected and a 32-residue segment corresponding to the filament framework could be identified in every case, supporting the notion that there is a common plan for the filament framework in all of these materials. The hairpin turns in the beta-sheet were also identified and shown to be unusually rich in proline residues and also of variable composition. Two variants of the mapping were found which have complimentary conformations of the hairpin turns and these are illustrated and discussed. Since feather keratin yields a fiber rather than a crystalline X-ray pattern refinement of the model is restricted to trial-and-error methods and the assumptions made in its derivation are critically examined and some possible modifications discussed.

Using structural data derived from crystal fragments of vimentin, three-dimensional models have been constructed for the major coiled-coil segments (1A, 1B and 2B) in epidermal and hair keratin intermediate filament molecules. Similarity and difference distributions arising from the heterodimer nature of the keratin molecules have been calculated, colour-coded for ease of observation and represented as movie clips. This approach has enabled the spatial distributions of the charged and apolar residues to be visualized along the seam between the chains and on the surface of the molecule, thus providing new insights into the features of the IF molecule that are important in assembly. An observation of note is that one face of both segment 1A and segment 1B is predominantly apolar and, furthermore, contains the bulk of the differences in the charged residues that occur between the two chains. The face rotated by 180 degrees contains far fewer apolar residues. This suggests the likely internal face of segments 1A and 1B and, hence, those sequence and spatial features that are important in assembly. In addition, the similarity distributions of the acidic and basic residues display a period of about 19 residues over much of each of the two faces of segment 1B. The two 19-residue periods are out of phase with respect to one another, however, thus leading to the previously recorded 9.51 residue period in the axial distributions of the acidic and the basic residues. The apparent doubling of the period arises because 9.51 residues corresponds to a non-integral number of turns of alpha-helical coiled coil.

Earlier studies established that substantial changes take place in the three-dimensional structure of the newly assembled trichocyte keratin intermediate filament (IF) during the oxidation process (Wang, H., Parry, D.A.D., Jones, L.N., Idler, W.W., Marekov, L.N., Steinert, P.M. 2000. In vitro assembly and structure of trichocyte keratin intermediate filaments: A novel role for stabilization by disulfide bonding. J. Cell Biol. 151, 1459-1468). The present contribution describes a re-examination of previous data in which more accurate values for the axial dispositions of the molecules have been obtained to yield the most detailed picture yet available of the structural changes that occur in vivo. In particular, it is shown that in the newly assembled (reduced) IF the crosslinking data are consistent with the detailed (8+0) model suggested earlier (Fraser, R.D.B., Parry, D.A.D. 2005. The three-dimensional structure of trichocyte (hard alpha-) keratin intermediate filaments: Features of the molecular packing deduced from the sites of induced crosslinks. J. Struct. Biol. 151, 171-181), in which eight four-chain protofilaments are arranged on an annular ring. For oxidized IF, however, the existing X-ray data require a periodic imperfection in the surface lattice which is substantial in the case of an (8+0) model and hence difficult to explain. In contrast, an alternative (7+1) model (Fraser, R.D.B., MacRae, T.P., Parry, D.A.D., Suzuki, E. 1986. Intermediate filaments in alpha-keratin. Proc. Natl. Acad. Sci. USA 83, 1179-1183) requires only a minor imperfection, and it is suggested that this is associated with the central protofilament. This suggestion is shown to be compatible with both the crosslinking data and a model for the axial distribution of electron density derived from the meridional X-ray pattern. In addition, evidence from an X-ray diffraction study of the follicle (Er Rafik, M., Briki, F., Burghammer, M., Doucet, J. 2006. In vivo formation of the hard alpha-keratin intermediate filament along a hair follicle: Evidence for structural polymorphism. J. Struct. Biol. 154, 79-88) and electron microscope studies of isolated reduced IF (Watts, N.R., Jones, L.N., Cheng, N., Wall, J.S., Parry, D.A.D., Steven, A.C. 2002. Cryo-electron microscopy of trichocyte (hard alpha-keratin) intermediate filaments reveals a low-density core. J. Struct. Biol. 137, 109-118) have been combined with earlier X-ray studies to give an estimate of the reduction in diameter that occurs in the IF due to the lateral reorganization of the protofilaments during the oxidation process. It has also been shown that the local coiled-coil geometry in the immediate vicinity of the contributing cysteine residues is necessarily disrupted, a feature consistent with the breadths of the near-equatorial layer lines in the X-ray diffraction pattern that indicate an average coherent length of coiled coil of only about 5 nm.

Intermediate filaments (IFs) represent one of the prominent cytoskeletal elements of metazoan cells. Their constituent proteins are coded by a multigene family, whose members are expressed in complex patterns that are controlled by developmental programs of differentiation. Hence, IF proteins found in epidermis differ significantly from those in muscle or neuronal tissues. Due to their fibrous nature, which stems from a fairly conserved central alpha-helical coiled-coil rod domain, IF proteins have long resisted crystallization and thus determination of their atomic structure. Since they represent the primary structural elements that determine the shape of the nucleus and the cell more generally, a major challenge is to arrive at a more rational understanding of how their nanomechanical properties effect the stability and plasticity of cells and tissues. Here, we review recent structural results of the coiled-coil dimer, assembly intermediates and growing filaments that have been obtained by a hybrid methods approach involving a rigorous combination of X-ray crystallography, small angle X-ray scattering, cryo-electron tomography, computational analysis and molecular modeling.

Sequence comparisons have been undertaken for all hair and epithelial keratin IF chains from a single species--human. The results lead to several new proposals. First, it is clear that not only is the chain structure of the molecule an obligate heterodimer but promiscuous association of Type I and Type II chains must occur in vivo. Second, the higher predicted content of alpha-helix in Type II chains in solution relative to that expected for Type I chains suggests that it is the Type II chains that precede their Type I counterparts and that they may serve as templates for molecule formation. Third, heterodimer formation leads naturally to greater structural and functional specificity, and this may be required not only because keratin IF have more interacting partners in its cell type than other types of IF have in theirs but also because hair and skin IF have two distinct structures that relate to the "reducing" or "oxidizing" environment in which they can find themselves. The transition between the two forms may require specific head/tail interactions and this, it is proposed, would be more easily accomplished by a heterodimer structure with its greater in-built specificity.

In this paper, we undertake a sequence analysis of the human keratin-associated proteins (KAP). This analysis has revealed two fundamental pentapeptide quasi-repeats (A and B) of the form C-C-X-P-X and C-C-X-S/T-S/T, respectively. The A repeats are also commonly found in two subforms A1 and A2, -C-C-Q-P-X and C-C-R-P-X, respectively-similar to those found in sheep wool 30-40 years previously. Some high-sulphur and ultra-high sulphur proteins contain predominantly A repeats or B repeats but not regular combinations of them, whereas others are characterised by a contiguous pair of pentapeptide repeats that largely (though imperfectly) alternate to generate decapeptide motifs of the form AB, A1B or A2B. The A and B repeats sometimes occur in complex runs and can generate both 19- and 20-residue repeats of the form BABB' or BA1AA, respectively, where the prime indicates a motif truncated by one residue. Likewise, a 42-residue repeat with BA1BXAAAB (40 residues) separated by a di-serine (two residues) has been observed in an ultra-high sulphur protein from cuticle. To understand the possible conformations adopted by the A and B motifs, a search was initiated of the PDB structural database for a number of overlapping pentapeptide repeats. The total number of matches was 658 and these were found in 451 different proteins. From representative and unique structures the means and standard deviations were calculated for the Phi(i) and Psi(i) angles for the C-C-X-P-X and the C-C-X-S/T-S/T motifs. Molecular modelling has been employed to represent the "average" structure found from crystallographic and nmr data determined for each motif in other proteins. The conformation of consecutive A repeats with proline residues in the cis state is akin to a string of disulphide bond-stabilised pentapeptide knots between which there is relative freedom of rotation about the single bonds that link them. For B pentapeptides, however, the likelihood that a similar disulphide bond is formed appears much lower. This may give additional conformational flexibility to the chain and hence allow the A pentapeptides greater opportunity to interact appropriately with the IF via disulphide bonds, ionic interactions and/or hydrogen bonding.

Recently, the spatial distribution of the crosslinks induced between lysine residues in trichocyte (alpha-) keratin intermediate filaments (IF) using disulfosuccinimidyl tartrate was analyzed in detail and the results used to provide information about the three-dimensional structure of the IF [Fraser, R.D.B., Parry, D.A.D., 2005. The three-dimensional structure of trichocyte (hard alpha-) keratin intermediate filaments: features of the molecular packing deduced from the sites of induced crosslinks. J. Struct. Biol. 151, 171-181.] The presence of small amounts of 0--> +/-4 crosslinkages between molecular strands four distant in the network implied that the three-dimensional network of interacting molecules must be deeply puckered, but no specific suggestions were made about the nature of the puckering. Whilst it was recognized that there may be more than one type of molecular environment in the structural repeat the initial analysis was confined to the simplest case in which all molecules had the same environment, that is to say the asymmetric unit comprised a single molecule. Further studies reported here suggest that it is likely that the asymmetric unit consists of at least two and possibly as many as four molecules and the implications of this for modeling the structure of trichocyte IF are discussed.

The conformation adopted by intermediate filament chains (IF) has been described in terms of a central rod domain with four, alpha-helical, left-handed coiled-coil segments (1A, 1B, 2A, and 2B) joined by linkers (L1, L12, and L2, respectively). The rod domain is terminated at its N- and C-terminal ends by "globular" head and tail domains, respectively. This analysis, initially undertaken about 20-25 years ago, was based on the recognition of an underlying heptad substructure in the sequence of the rod domain, the presence of which can be directly associated with an alpha-helical coiled-coil structure. In this work, a hendecad sequence motif that is closely related to the heptad repeat but which is nonetheless significantly different from it has been recognized in the primary structure of segments 2A and linker L2. This motif, which is 11 residues long and structurally equivalent to a true heptad plus another heptad with an inclusive stutter, is consistent with the chains adopting a continuous right-handed coiled-coil structure with a long-period pitch length. It is therefore predicted that segment 2 as a whole may have a coiled-coil conformation with both right-handed (2A+L2) and left-handed (2B) regions. The changeover in handedness would be expected to occur at the C-terminal end of linker L2 and N-terminal end of segment 2B.

Keratins are intermediate filament-forming proteins that provide mechanical support and fulfill a variety of additional functions in epithelial cells. In 1982, a nomenclature was devised to name the keratin proteins that were known at that point. The systematic sequencing of the human genome in recent years uncovered the existence of several novel keratin genes and their encoded proteins. Their naming could not be adequately handled in the context of the original system. We propose a new consensus nomenclature for keratin genes and proteins that relies upon and extends the 1982 system and adheres to the guidelines issued by the Human and Mouse Genome Nomenclature Committees. This revised nomenclature accommodates functional genes and pseudogenes, and although designed specifically for the full complement of human keratins, it offers the flexibility needed to incorporate additional keratins from other mammalian species.

The beta-form of protein folding, one of the earliest protein structures to be defined, was originally observed in studies of silks. It was then seen in early studies of synthetic polypeptides and, of course, is now known to be present in a variety of guises as an essential component of globular protein structures. However, in the last decade or so it has become clear that the beta-conformation of chains is present not only in many of the amyloid structures associated with, for example, Alzheimer's Disease, but also in the prion structures associated with the spongiform encephalopathies. Furthermore, X-ray crystallography studies have revealed the high incidence of the beta-fibrous proteins among virulence factors of pathogenic bacteria and viruses. Here we describe the basic forms of the beta-fold, summarize the many different new forms of beta-structural fibrous arrangements that have been discovered, and review advances in structural studies of amyloid and prion fibrils. These and other issues are described in detail in later chapters.

Objective: The objective was to determine the types of organisms which cause acute otitis media with a tympanostomy tube and to ascertain their frequency distribution., Study Design and Setting: Prospective, randomized, multi-institutional clinical trials. Both private and academic sites were included., Results: 1309 isolates were recovered from 956 draining ears. Streptococcus pneumonia was recovered from 17%, Staphylococcus aureus from 13%, H flu from 18% and Pseudomonas aeruginosa from 12%. Fungal organisms were recovered from 5% of total isolates and 4% from single isolates., Conclusions: AOMT is microbiologically different than AOM with an intact TM. There is no evidence that resistance develops as result of topical treatment., Significance: The study demonstrates that AOMT is frequently caused by organisms not susceptible to oral antibiotics approved for children, but which are sensitive to topical ear drops.

In Drosophila melanogaster, X chromosome dosage compensation is achieved by doubling the transcription of most X-linked genes. The male-specific lethal (MSL) complex is required for this process and binds to hundreds of sites on the male X chromosome. The MSL1 protein is essential for X chromosome binding and serves as a central scaffold for MSL complex assembly. We find that the amino-terminal region of MSL1 binds to hundreds of sites on the X chromosome in normal males but only to approximately 30 high-affinity sites in the absence of endogenous MSL1. Binding to the high-affinity sites requires a basic motif at the amino terminus that is conserved among Drosophila species. X chromosome binding also requires a conserved leucine zipper-like motif that binds to MSL2. A glycine-rich motif between the basic and leucine-zipper-like motifs mediates MSL1 self-association in vitro and binding of the amino-terminal region of MSL1 to the MSL complex assembled on the male X chromosome. We propose that the basic region may mediate DNA binding and that the glycine-rich region may promote the association of MSL complexes to closely adjacent sites on the X chromosome.

Background & Aims: Keratins 8 and 18 protect the liver from stress. Keratin 8 and 18 variants in 17 of 467 liver disease explants and 2 of 349 blood bank controls were previously reported in 5 analyzed exonic regions. We asked whether mutations were present in the remaining 10 exons of keratins 8 and 18., Methods: Exonic regions were polymerase chain reaction-amplified from genomic DNA, isolated from the above-mentioned 2 cohorts, and analyzed for the presence of mutations. Mutant keratins were also studied biochemically., Results: We identified 10 novel keratin 8 and 18 heterozygous variants in 44 of 467 explants and 11 of 349 controls: keratin 18 deletion (delta64-71), a keratin 8 frameshift that truncates the last 14 amino acids; 8 missense keratin 8 and 18 alterations; and several new polymorphisms. The most common variant, keratin 8 R340H, at the highly conserved R340 was found in 30 of 467 explants and 10 of 349 controls (P = .02) and was confirmed in the diseased livers by generation of an R340H-specific antibody. Germline transmission and variant protein expression were verified. The mutations involved a variety of liver diseases, and some variants had an ethnic background preponderance. Mutations that introduced disulfide bonds (keratin 8 G61C or R453C) decreased keratin solubility, particularly after oxidative stress, whereas others decreased keratin 8 phosphorylation (keratin 8 G433S)., Conclusions: The overall frequency of keratin 8 and 18 variants was 12.4% in 467 liver disease explants and 3.7% in 349 blood bank controls (P < .0001). Variants can alter keratin solubility or phosphorylation and may render individuals susceptible to end-stage liver disease, depending on their genetic background and exposure to other insults, such as alcohol or viral infection.

Objectives: To compare magnetic resonance imaging (MRI) to high-resolution computed tomography (HRCT) in the preoperative evaluation of pediatric cochlear implant candidates., Methods: The charts of pediatric cochlear implant candidates evaluated between July 1, 2000 and November 30, 2003 with an MRI scan of the inner ear were included in the study. Fifty-six patients were included. Associated HRCT scans were examined. Abnormalities of the cochlea, cochlear nerve, endolymphatic sac, endolymphatic duct, vestibule, and modiolus were noted. A pediatric neuroradiologist gave an opinion as to whether patients with anomalies seen with MRI but without associated HRCT would have been identified by HRCT., Results: Of the 112 temporal bones imaged with MRI, the following abnormalities were encountered: 32% (36/112) had abnormalities of the cochlear turns, 30% (34/112) had abnormal signal in the modiolus, 23% (26/112) had abnormal vestibulae, 16% (18/112) had abnormal endolymphatic ducts, 15% (17/112) had abnormal endolymphatic sacs, 12% (13/112) had abnormalities of the cochlear nerves, 29% (17/56) had abnormalities of the brain. HRCT cannot directly evaluate the cochlear nerve. Available HRCT findings were combined with radiologic opinion and compared with MRI findings. The percentages of abnormalities identifiable by HRCT when compared with those seen with MRI are cochlea 42% (15/36), modiolus 35% (12/34), vestibulae 88% (23/26), endolymphatic duct 100% (18/18), and endolymphatic sac 6% (1/17)., Conclusion: MRI is more sensitive and specific in diagnosing soft tissue abnormalities in the inner ear than HRCT in cochlear implant candidates (Fig. 4). Moreover, the abnormalities detected with MRI are more likely to influence the implantation process (e.g., asymmetric nerve aplasia, cochlear obstruction). (Figure is included in full-text article).

MACF1 (microtubule actin crosslinking factor), also called ACF7 (actin crosslinking family 7) is a cytoskeletal linker protein that can associate with both actin filaments and microtubules. We have identified a novel alternatively spliced isoform of MACF1. We named this isoform MACF1b and renamed the original isoform MACF1a. MACF1b is identical to MACF1a, except that it has a region containing plakin (or plectin) repeats in the middle of the molecule. MACF1b is ubiquitously expressed in adult tissues with especially high levels in the lung. We studied the subcellular localization of MACF1b proteins in mammalian cell lines. In two lung cell lines, MACF1b was chiefly localized to the Golgi complex. Upon treatments that disrupt the Golgi complex, MACF1b redistributed into the cytosol, but remained co-localized with the dispersed Golgi ministacks. MACF1b proteins can be detected in the enriched Golgi fraction by western blotting. The domain of MACF1b that targets it to the Golgi was found at the N-terminal part of the region that contains the plakin repeats. Reducing the level of MACF1 proteins by small-interfering RNA resulted in the dispersal of the Golgi complex.

The spatial distribution of the crosslinks that can be induced between lysine residues in trichocyte (alpha-) keratin intermediate filaments (IF) using disulfosuccinimidyl tartrate has been analyzed in detail and the results used to provide information about the three-dimensional (3-D) structure. The pattern of inter-molecular interactions derived from earlier studies is essentially two-dimensional in that it involves projection on to a cylinder followed by unwrapping to give a sheet. Crosslinks are observed between molecular strands four apart and it is shown that this can only occur if the paths of the molecular strands through the IF are systematically distorted. These crosslinks are clustered axially at intervals of around 15 nm, a value closely related to the pitch length of the constituent coiled-coil molecules in the rod domains. The number of crosslinks between adjacent molecular strands shows a striking difference depending on lateral direction and provides support for the concept of a head-to-tail stacking of tetramers defined by the A(CN) mode of packing to form protofilament substructures in the fully formed IF. Each protofilament would consist of a pair of oppositely directed molecular strands stabilized by A(11) and A(22) interactions identified in earlier work. A detailed model for the IF in the reduced state comprising a ring of eight protofilaments is suggested. When combined with earlier studies of crosslink formation in the oxidized state, the present findings lead to the conclusion that there is a major reorganization of the molecular packing within the protofilaments during keratinization in vivo. Taken in conjunction with existing X-ray data on the fully keratinized structures, the new evidence for a protofilament substructure also enables a detailed 3-D model for the mature IF to be suggested.

The amino acid sequences of increasingly large proteins have been determined in recent years, and it has become more and more apparent that within these sequences nature has employed only a finite number of structural?functional motifs. These may be strung along the sequence in tandem and, in some cases, several hundred times. In other instances, the positions of the motifs show little obvious order as regards to their relative linear arrangement within the sequence. The observed sequence repeats have been shown to vary in size over at least two orders of magnitude. It is shown here that the repeats can readily be classified on the basis of character, and five distinct groups have been identified. The first of these (Type A) represents those motifs that are fixed in length and conserved absolutely in sequence (>99%); the second (Type B) includes motifs that are also fixed in length, but where absolute sequence conservation occurs only in some positions of the repeat. The third category (Type C) contains fixed length motifs, but the character of only some of the positions in the motif is maintained. The fourth group (Type D) includes motifs that have nonintegral lengths. The fifth class (Type E) contains motifs, often displaying some variations in their lengths even within a single species, which maintain a discrete structural form related directly to their function. Examples are presented for each category of repeat, and these are drawn almost exclusively from the fibrous proteins and those proteins that are normally associated with them in vivo.

A large number of intermediate filament (IF) chains have now been sequenced. From these data, it has been possible to deduce the main elements of the secondary structure, especially those lying within the central rod domain of the molecule. These conclusions, allied to results obtained from crosslinking studies, have shown that at least four unique but related structures are adopted by the class of structures known generically as intermediate filaments: (1) epidermal and reduced trichocyte keratin; (2) oxidized trichocyte keratin; (3) desmin, vimentin, neurofilaments, and related Type III and IV proteins; and (4) lamin molecules. It would be expected that local differences in sequences of the proteins in these four groups would occur, and that this would ultimately relate to assembly. Site-directed mutagenesis and theoretical methods have now made it possible to investigate these ideas further. In particular, new data have been obtained that allow the role played by some individual amino acids or a short stretch of sequence to be determined. Among the observations catalogued here are the key residues involved in intra- and interchain ionic interactions, as well as those involved in stabilizing some modes of molecular aggregation; the structure and role of subdomains in the head and tail domains; the repeat sequences occurring along the length of the chain and their structural significance; trigger motifs in coiled-coil segments; and helix initiation and termination motifs that terminate the rod domain. Much more remains to be done, not least of which is gaining an increased understanding of the many subtle differences that exist between different IF chains at the sequence level.

Coiled-coil proteins, collagen, and elastomers together comprise an important subset of the fibrous proteins. The former group-the alpha-fibrous coiled-coil proteins-are widely distributed in nature and, indeed, the characteristic heptad motif has been recognized as an oligomerisation motif in fibril-forming collagens. This volume has selected a number of the alpha-fibrous proteins for detailed discussion, including intermediate filament proteins, the spectrin superfamily, and fibrin?fibrinogen. Of particular interest is the growing realization that the design principles governing the structures of these coiled-coil proteins are now largely discernible and can be specified with a high degree of confidence, due in large part to the wealth of crystal structure data now available. Within the connective tissues covered in this volume, two constituents of defining importance mechanically are the collagen fibrils?networks and the elastic fibers. Crystal structures of collagen peptides have been published and are described. The effects of the precise sequence of the distinct constituent triplets on molecular conformation have also become clearer. The ultrastructures of connective tissues are largely defined by the spatial arrangement of the collagen fibrils and networks, and this is elucidated here in some detail. The elastic fibers with their elastin cores and fibrillin-containing microfibril palisades are also described. A theme underlying all of the proteins discussed in this volume is the significantly increased effort to characterize the structures and functions of mutants. Some of these occur naturally and lead to various disease states, while others have been genetically engineered in order to study design principles.

Detailed analyses of protein structures provide an opportunity to understand conformation and function in terms of amino acid sequence and composition. In this work, we have systematically analyzed the characteristic features of the amino acid residues found in alpha-helical coiled-coils and, in so doing, have developed indices for their properties, conformational parameters, surrounding hydrophobicity and flexibility. As expected, there is preference for hydrophobic (Ala, Leu), positive (Lys, Arg) and negatively (Glu) charged residues in coiled-coil domains. However, the surrounding hydrophobicity of residues in coiled-coil domains is significantly less than that for residues in other regions of coiled-coil proteins. The analysis of temperature factors in coiled-coil proteins shows that the residues in these domains are more stable than those in other regions. Further, we have delineated the medium- and long-range contacts in coiled-coil domains and compared the results with those obtained for other (non-coiled-coil) parts of the same proteins and non-coiled-coil helical segments of globular proteins. The residues in coiled-coil domains are largely influenced by medium-range contacts, whereas long-range interactions play a dominant role in other regions of these same proteins as well as in non-coiled-coil helices. We have also revealed the preference of amino acid residues to form cation-pi interactions and we found that Arg is more likely to form such interactions than Lys. The parameters developed in this work can be used to understand the folding and stability of coiled-coil proteins in general.

The sequence of a protein chain determines both its conformation and its function in vivo. An attempt is made to gain an understanding of the classes of deformations that can arise in an important structural motif, the alpha-helical coiled coil, as a consequence of mutations occurring in its underlying heptad substructure. In order to do so we consider the model structure of segment 1A in intermediate filaments and then investigate the structures arising from each of the 22 mutations observed in cytokeratin K5/K14 molecules that lead to variants of epidermolysis bullosa simplex. These are refined separately using a molecular dynamics protocol. The mutations often result in a significant distortion of the backbone over a turn or so of the alpha helix in either the chain itself or its constituent partner, leading to the likelihood of impaired chain aggregation and hence molecular assembly. One mutant (K14-L143P; 1A-28) gave rise to structural distortion along almost the entire length of segment 1A. The remaining structures showed less deformation, and normal-looking intermediate filaments are likely in vivo. In addition, an identical mutation in the same position in each of the chains in the heterodimer did not necessarily give equivalent structural distortions. Although proline mutations frequently lead to the most severe structural deformations, a non-proline substitution (K14-R125S; 1A-10) gave rise to the largest local structural disruption that was observed. Unexpectedly, mutations in positions a and d were not always of the greatest structural significance, although three in position a were shown by AGADIR to result in a significant increase in alpha-helix stability., (Copyright 2004 Wiley-Liss, Inc.)

Pseudofolliculitis barbae (PFB) is a common hair disorder characterized by a pustular foreign body inflammatory reaction that is induced by ingrown hairs of the facial and submental (barbea) regions after regular shaving. It occurs predominantly in black males, while it is rather rare and usually far less severe in Caucasian males. Black individuals have a higher propensity of developing PFB due to their genetic predisposition for curly hair which inherently possesses a much higher risk of growing back into the skin than straight or wavy hair. The PFB process is, however, not gender dependent nor restricted to the face, but can occur in any skin region once regular shaving, plucking, or other traumatic means of hair removal are instituted. Through a family study and a large-scale investigation of randomly sampled PFB-affected and -unaffected individuals, this study demonstrates that an unusual single-nucleotide polymorphism, which gives rise to a disruptive Ala12Thr substitution in the 1A alpha-helical segment of the companion layer-specific keratin K6hf of the hair follicle, is partially responsible for the phenotypic expression and represents an additional genetic risk factor for PFB.

Alpha-helical coiled-coils are widely occurring protein oligomerization motifs. Here we show that most members of the collagen superfamily contain short, repeating heptad sequences typical of coiled coils. Such sequences are found at the N-terminal ends of the C-propeptide domains in all fibrillar procollagens. When fused C-terminal to a reporter molecule containing a collagen-like sequence that does not spontaneously trimerize, the C-propeptide heptad repeats induced trimerization. C-terminal heptad repeats were also found in the oligomerization domains of the multiplexins (collagens XV and XVIII). N-terminal heptad repeats are known to drive trimerization in transmembrane collagens, whereas fibril-associated collagens with interrupted triple helices, as well as collagens VII, XIII, XXIII, and XXV, were found to contain heptad repeats between collagen domains. Finally, heptad repeats were found in the von Willebrand factor A domains known to be involved in trimerization of collagen VI, as well as in collagen VII. These observations suggest that coiled-coil oligomerization domains are widely used in the assembly of collagens and collagen-like proteins.

The in vitro polymerization and tissue-specific expression patterns of the four essential intermediate filament (IF) proteins (A1, A2, A3, B1) and the non-essential IF protein A4 were analyzed. Recombinant B1, used as a probe in blot overlay assays of the 11 Caenorhabditis elegans IF proteins, reacted strongly with proteins A1 to A4, indicating a heterotypic interaction. Obligate heteropolymeric filament assembly in vitro was confirmed by electron microscopy. Protein B1 formed long IF when mixed with an equimolar amount of A1, A2 or A3. Developmentally regulated coexpression of B1 and one or more members of the A family was found with GFP-promoter reporters. This coexpression pattern argues for a heteropolymer system in vivo. One or both splice variants of the B1 gene are always coexpressed in a tissue-specific manner with at least one member of the A family in hypodermis, pharynx, pharyngeal-intestinal valve, excretory cells, uterus, vulva and rectum. Interestingly, while the intestine normally lacks a B1/A pair, the dauer larva shows intestinal B1 and A4. These results are in line with similar postembryonic phenotypes of the hypodermis induced by RNA interference (RNAi) of genes B1, A2 and A3. Similarly, defects of the pharynx and its A1-GFP containing tonofilaments observed in the postembryonic B1 RNAi phenotype are consistent with the coexpression of B1 and A1 in the marginal cells. Thus RNAi analyses provide independent evidence for the existence of the B1/A obligate heteropolymer system in vivo. Proteins A1 and B1 have a similar and rather slow turnover rate in photobleaching experiments of the pharynx tonofilaments.

Trichohyalin is expressed in specialized epithelia that are unusually mechanically strong, such as the inner root sheath cells of the hair follicle. We have previously shown that trichohyalin is sequentially subjected to post-synthetic modifications by peptidylarginine deaminases, which convert many of its arginines to citrullines, and by transglutaminases, which introduce intra- and interprotein chain cross-links. Here we have characterized in detail the proteins to which it becomes cross-linked in vivo in the inner root sheath of the mouse hair follicle. We suggest that it has three principal roles. First, it serves as an interfilamentous matrix protein by becoming cross-linked both to itself and to the head and tail end domains of the inner root sheath keratin intermediate filament chains. A new antibody reveals that arginines of the tail domains of the keratins are modified to citrullines before cross-linking, which clarifies previous studies. Second, trichohyalin serves as a cross-bridging reinforcement protein of the cornified cell envelope of the inner root sheath cells by becoming cross-linked to several known or novel barrier proteins, including involucrin, small proline-rich proteins, repetin, and epiplakin. Third, it coordinates linkage between the keratin filaments and cell envelope to form a seamless continuum. Together, our new data document that trichohyalin is a multi-functional cross-bridging protein that functions in the inner root sheath and perhaps in other specialized epithelial tissues by conferring to and coordinating mechanical strength between their peripheral cell envelope barrier structures and their cytoplasmic keratin filament networks.

The intermediate filaments (IF) in trichocyte (hard-alpha) keratin form ordered aggregates that are infiltrated by sulfur-rich and tyrosine-rich proteins during fiber development to give a filament-matrix texture, which is stabilized in the later stages by the formation of disulfide linkages. Two polymorphic forms of macrofibril are found in the cortical cells of fine Merino wool. In the first the packing of the IF in the macrofibril is quasi-hexagonal whilst in the second the IF are packed in cylindrical sheets around a central core. In hairs the second type generally predominate. In the present contribution specific models for the mechanisms of nucleation and growth are developed for the two types of macrofibril and their applicability tested by analyzing transmission electron micrographs of wool and hair. Evidence is presented which supports the idea that sheet formation plays an important role in both types of macrofibril assembly and it is suggested that differing intersheet interactions are responsible for the differences between the ortho- and para-types. It is shown that the increase in IF tilt with radius in the ortho-type can be related to the surface lattice of the IF as determined from X-ray diffraction studies. Two possible types of intersheet interaction in the ortho-type are discussed, the first leading to an increase of around 0.5 degrees in IF tilt per layer and the second leading to a much larger tilt of 9.4 degrees per layer. A crude estimate based on the decrease in visibility of the IF with increasing radius in cross-section yielded a value of 0.35 degrees -0.7 degrees.

The so-called hard alpha-keratins, such as quill and hair, have a composite structure in which intermediate filaments (IF) are embedded in a sulfur-rich matrix. Recent studies of these trichocyte keratin IF have revealed that substantial changes in the molecular architecture take place when oxidation of the cysteine residues occurs as part of the terminal differentiation/keratinization process. Recent cryoelectron microscope studies suggest that the IF has a tubular structure prior to keratinization, but transmission electron micrographs of thin sections of fully keratinized fibers exhibit a "ring-core" structure. In the present contribution we develop a generic model for the IF in the reduced state based on cross-linking studies and discuss two possibilities for the way in which this structure may be modified during the keratinization process.

Early electron microscope studies of developing wool and hair established that trichocyte (hard alpha-) keratin fibers have a composite structure in which filaments, subsequently shown to belong to the class of intermediate filaments (IF), were embedded in a matrix of sulfur-rich proteins. These studies also showed that the IF aggregate in a variety of ways to form what have been termed macrofibrils. Assembly into sheets appears to be an important initial factor in aggregation, and in the present contribution the structural principles governing sheet formation are formulated and specific models for the interaction between neighboring IF in a sheet are proposed, based on existing X-ray diffraction, electron microscope, and crosslinking data. All of the trichocyte keratins so far examined by electron microscopy exhibit similar filament/matrix textures and the mechanism of sheet formation proposed here is likely to have general applicability.

Attempts at predicting the relative axial alignments of fibrous protein molecules in filamentous structures have relied upon representing the (multichain) molecular structure by a one-dimensional sequence of amino acids. Potential intermolecular ionic and apolar interactions were counted and determined as a function of the relative axial stagger between the molecules. No attempts were made to consider the azimuthal aspect of the interacting molecules and neither were apolar or ionic energy terms used. Surprisingly, this simple approach proved remarkably informative and yielded accurate predictions of the axial periods present. However, a more comprehensive analysis involving the energetics of aggregation taking due regard for the relative azimuths of the molecules as well as their separation should decrease the noise level in the calculations and reveal other pertinent information. Toward that end, we have modeled the interaction between two alpha-helical coiled-coil segments in intermediate filament molecules (1B segments from human vimentin). The relative axial alignment and polarity of the molecules is already known from detailed crosslinking studies and this provides a criterion against which the success (or otherwise) of the modeling can be judged. The results confirm that an antiparallel alignment of two 1B segments is preferred over any of the parallel options (as observed experimentally). The calculated axial alignment, however, is not identical to that observed from detailed crosslinking studies indicating that other parts of the molecule (probably the head and tail domains as well as other coiled-coil segments) have a crucial role in determining the precise mode of axial aggregation. The results also show that the apolar interactions seem to be significantly less important in the alignment process than the ionic ones. This is consistent with the observation of a well-defined period in the linear disposition of the charged (but not apolar) residues along the length of the outer surface of the vimentin molecule., (Copyright 2002 Wiley-Liss, Inc.)