Denise B. Flaherty

Tyrosine cross-linking of extracellular matrix is catalyzed by Duox, a multidomain oxidase/peroxidase with homology to the phagocyte oxidase subunit gp91phox

High molecular weight homologues of gp91phox, the superoxide-generating subunit of phagocyte nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase, have been identified in human (h) and Caenorhabditis elegans (Ce), and are termed Duox for “dual oxidase” because they have both a peroxidase homology domain and a gp91phox domain. A topology model predicts that the enzyme will utilize cytosolic NADPH to generate reactive oxygen, but the function of the ecto peroxidase domain was unknown. Ce-Duox1 is expressed in hypodermal cells underlying the cuticle of larval animals. To investigate function, RNA interference (RNAi) was carried out in C. elegans. RNAi animals showed complex phenotypes similar to those described previously in mutations in collagen biosynthesis that are known to affect the cuticle, an extracellular matrix. Electron micrographs showed gross abnormalities in the cuticle of RNAi animals. In cuticle, collagen and other proteins are cross-linked via di- and trityrosine linkages, and these linkages were absent in RNAi animals. The expressed peroxidase domains of both Ce-Duox1 and h-Duox showed peroxidase activity and catalyzed cross-linking of free tyrosine ethyl ester. Thus, Ce-Duox catalyzes the cross-linking of tyrosine residues involved in the stabilization of cuticular extracellular matrix.

Phosphorylation of human tau protein by microtubule-associated kinases: GSK3? and cdk5 are key participants

Microtubules (MTs), primarily composed of α and β tubulin polymers, must often work in concert with microtubule‐associated proteins (MAPs) in order to modulate their functional demands. In a mature brain neuron, one of the key MAPs that resides primarily in the axonal compartment is the tau protein. Tau, in the adult human brain, is a set of six protein isoforms, whose binding affinity to MTs can be modulated by phosphorylation. In addition to the role that phosphorylation of tau plays in the “normal” physiology of neurons, hyperphosphorylated tau is the primary component of the fibrillary pathology in Alzheimers disease (AD). Although many protein kinases are known to phosphorylate tau in vitro, the in vivo players contributing to the hyperphosphorylation of tau remain elusive. The experiments in this study attempt to define which protein kinases and protein phosphatases reside in the associated network of microtubules, thereby being strategically positioned to influence the phosphorylation of tau. Microtubule fractions are utilized to determine which of the microtubule‐associated kinases most readily impacts the phosphorylation of tau at “AD‐like” sites. Results from this study indicate that PKA, CK1, GSK3β, and cdk5 associate with microtubules. Among the MT‐associated kinases, GSK3β and cdk5 most readily contribute to the ATP‐induced “AD‐like” phosphorylation of tau. J. Neurosci. Res. 62:463–472, 2000.

Nuclear titin interacts with A- and B-type lamins in vitro and in vivo

Lamins form structural filaments in the nucleus. Mutations in A-type lamins cause muscular dystrophy, cardiomyopathy and other diseases, including progeroid syndromes. To identify new binding partners for lamin A, we carried out a two-hybrid screen with a human skeletal-muscle cDNA library, using the Ig-fold domain of lamin A as bait. The C-terminal region of titin was recovered twice. Previous investigators showed that nuclear isoforms of titin are essential for chromosome condensation during mitosis. Our titin fragment, which includes two regions unique to titin (M-is6 and M-is7), bound directly to both A- and B-type lamins in vitro. Titin binding to disease-causing lamin A mutants R527P and R482Q was reduced 50%. Studies in living cells suggested lamin-titin interactions were physiologically relevant. In Caenorhabditis elegans embryos, two independent C. elegans (Ce)-titin antibodies colocalized with Ce-lamin at the nuclear envelope. In lamin-downregulated [lmn-1(RNAi)] embryos, Ce-titin was undetectable at the nuclear envelope suggesting its localization or stability requires Ce-lamin. In human cells (HeLa), antibodies against the titin-specific domain M-is6 gave both diffuse and punctate intranuclear staining by indirect immunofluorescence, and recognized at least three bands larger than 1 MDa in immunoblots of isolated HeLa nuclei. In HeLa cells that transiently overexpressed a lamin-binding fragment of titin, nuclei became grossly misshapen and herniated at sites lacking lamin B. We conclude that the C-terminus of nuclear titin binds lamins in vivo and might contribute to nuclear organization during interphase.

Titins in C. elegans with Unusual Features: Coiled-coil Domains, Novel Regulation of Kinase Activity and Two New Possible Elastic Regions

We report that there are previously unrecognized proteins in Caenorhabditis elegans that are similar to the giant muscle proteins called titins, and these are encoded by a single approximately 90kb gene. The gene structure was predicted by GeneMark.hmm and then experimentally verified. The Ce titin gene encodes polypeptides of 2.2MDa, 1.2MDa and 301kDa. The 2.2MDa isoform resembles twitchin and UNC-89 in that it contains multiple Ig (56) and FnIII (11) domains, and a single protein kinase domain. In addition, however, the 2.2MDa isoform contains four classes of short, 14-51 residue, repeat motifs arranged mostly in many tandem copies. One of these tandem repeat regions is similar to the PEVK regions of vertebrate and fly titins. As the PEVK region is one of the main elastic elements of the titins and is also composed of short tandem repeats, this suggests that the repeat motifs in the Ce titins may have a similar elastic function. An interesting aspect of the two largest Ce titin isoforms, is that in contrast to other members of the twitchin/titin family, there are multiple regions which are likely to form coiled-coil structure. In transgenic animals, the first approximately 100 residues of the largest isoforms targets to dense bodies, the worm analogs of Z-discs. Anti-Ce titin antibodies show localization to muscle I-bands beginning at the L2-L3 larval stages and this pattern continues into adult muscle. Ce titins may not have a role in early myofibril assembly: (1) Ce titins are too short to span half a sarcomere, and the onset of their expression is well after the initial assembly of thick filaments. (2) Ce titins are not localized to I-bands in embryonic or L1 larval muscle. The Ce titin protein kinase domain is most similar to the kinase domains of the twitchins and projectin. The Ce titin kinase has protein kinase activity in vitro, and this activity is regulated by a novel mechanism.

Titin/connectin-related proteins in C. elegans: a review and new findings.

Much is still unknown about mechanisms by which myofibrils assemble from their components, and how these precise structures are maintained in the face of repeated muscle activity. A number of labs, including ours, are exploiting the ability to analyze mutants in the nematode C. elegans to obtain insights into these questions. C. elegans is a particularly attractive organism in which to study muscle (Waterston, 1988; Moerman and Fire, 1997). Sophisticated forward, and more recently, reverse genetic approaches are available to reveal phenotypes. The usual self-fertilization allows propagation of muscle mutants that render animals so uncoordinated that they would be unable to mate. Optical transparency allows evaluation of muscle structure by polarized light microscopy. The muscles used for locomotion reside in the body wall (Figure 1). In the adult, there are 95 spindle shaped mononuclear cells divided amongst four quadrants just underlying a basement membrane, hypodermis and cuticle. In each quadrant, the cells are arranged in interlocking pairs. By polarized light microscopy, obvious striations are seen; bright (birefringent) A-bands alternate with dark I-bands; each Iband contains a row of dense bodies, which are the analogs of Z-discs in vertebrate striated muscle. Because the striations lie at a slightly oblique angle with respect to the long axis of the animal, this muscle is called ‘obliquely striated’. In contrast to vertebrate striated muscle, in which myofibrils fill the entire cell, in nematode body wall muscle, the equivalent myofilament lattice is restricted to a narrow zone of 1.5 lm on the outer side of the cell, just beneath the cell membrane. The thin filaments are attached to the dense bodies, and the thick filaments are organized around M-lines. All the dense bodies and M-lines are anchored to the muscle cell membrane, which is attached to the hypodermis and cuticle. This allows the force of muscle contraction to be transmitted directly to the cuticle and allows movement of the whole animal. Thus, worm muscle M-lines and dense bodies serve the function of analogous structures in vertebrate muscle. But, in addition, because of their membrane anchorage and protein composition, they are also similar to vertebrate non-muscle focal adhesions. The second largest set of muscle resides in the pharynx, the neuromuscular pump near the front of the worm used for pumping-in and grinding-up bacteria before passage into the intestine. Studies during the past 30 years in over a dozen labs have defined many components of C. elegans myofibrils and their membrane-extracellular matrix attachment structures. Most of these proteins were first defined through mutations – most falling into one of two phenotypic classes. In one class, the uncoordinated or ‘Unc’ class, animals are slow moving or paralyzed as adults. The second class, the ‘Pat’ class of mutants (paralyzed arrested at two-fold) have a characteristic embryonic lethality in which embryos do not move within the eggshell and stop development at the twofold stage (Williams and Waterston, 1994). A few genes have loss-of-function Unc and null Pat phenotypes; examples include unc-97 (PINCH) (Hobert et al., 1999) and unc-45 (myosin chaperone, UCS family member) (Barral et al., 1998). To date, five sets of proteins have been shown to be members of the intracellular, mostly muscle branch of the Ig superfamily in C. elegans: (1) twitchin, (2) UNC-89, (3) Ce titin, (4) DIM-1 (Rogalski et al., 2003), and (5) kettin. This review will focus on our current understanding of twitchin, UNC-89 and Ce titin, as these have been, or are currently, being studied in our laboratory. Kettin has been well studied in Drosophila, Lethocerus and crayfish. Nematode kettin was first described by Hakeda et al. (2000) and Kolmerer et al. (2000), and is 4000 residues long containing 31 Ig domains, most of which are separated by a conserved spacer sequence of 35 residues. Further information and new data on C. elegans kettin appear in an article by S. Ono, elsewhere in this volume.

Extensive and modular intrinsically disordered segments in C. elegans TTN-1 and implications in filament binding, elasticity and oblique striation.

TTN-1, a titin like protein in Caenorhabditis elegans, is encoded by a single gene and consists of multiple Ig and fibronectin 3 domains, a protein kinase domain and several regions containing tandem short repeat sequences. We have characterized TTN-1s sarcomere distribution, protein interaction with key myofibrillar proteins as well as the conformation malleability of representative motifs of five classes of short repeats. We report that two antibodies developed to portions of TTN-1 detect an approximately 2-MDa polypeptide on Western blots. In addition, by immunofluorescence staining, both of these antibodies localize to the I-band and may extend into the outer edge of the A-band in the obliquely striated muscle of the nematode. Six different 300-residue segments of TTN-1 were shown to variously interact with actin and/or myosin in vitro. Conformations of synthetic peptides of representative copies of each of the five classes of repeats--39-mer PEVT, 51-mer CEEEI, 42-mer AAPLE, 32-mer BLUE and 30-mer DispRep--were investigated by circular dichroism at different temperatures, ionic strengths and solvent polarities. The PEVT, CEEEI, DispRep and AAPLE peptides display a combination of a polyproline II helix and an unordered structure in aqueous solution and convert in trifluoroethanol to alpha-helix (PEVT, CEEEI, DispRep) and beta-turn (AAPLE) structures, respectively. The octads in BLUE motifs form unstable alpha-helix-like structures coils in aqueous solution and negligible heptad-based, alpha-helical coiled-coils. The alpha-helical structure, as modeled by threading and molecular dynamics simulations, tends to form helical bundles and crosses based on its 8-4-2-2 hydrophobic helical patterns and charge arrays on its surface. Our finding indicates that APPLE, PEVT, CEEEI and DispRep regions are all intrinsically disordered and highly reminiscent of the conformational malleability and elasticity of vertebrate titin PEVK segments. The proposed presence of long, modular and unstable alpha-helical oligomerization domains in the BLUE region of TTN-1 could bundle TTN-1 and stabilize oblique striation of the sarcomere.

Regulation of Tau Phosphorylation in Microtubule Fractions by Apolipoprotein E

In Alzheimers disease (AD), a family of proteins collectively named tau are displaced from their normal association with microtubules and are found in in a hyperphosphorylated state deposited into paired helical filaments (PHFs). PHFs are the hallmark cytoskeletal pathology of the disease, and the degree of PHF pathology correlates with the clinical severity of AD. Certain apolipoprotein E (apoE) isoforms have been identified as either risk or protective factors for AD, and one of the proposed mechanisms involves an interaction and potentially modulatory effects on tau hyperphosphorylation by the different apoE isoforms. In these studies, we directly tested the effects of apoE, E2 ,E3, and E4 on AD‐like phosphorylation of tau in brain microtubule fractions. We found that apoE attenuates tau hyperphosphorylation in the fractions, but the pattern was indistinguishable for the different isoforms. J. Neurosci. Res. 56:271–274, 1999.