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.

The C-terminal tail of UNC-60B (actin depolymerizing factor/cofilin) is critical for maintaining its stable association with F-actin and is implicated in the second actin-binding site.

Actin depolymerizing factor (ADF)/cofilin changes the twist of actin filaments by binding two longitudinally associated actin subunits. In the absence of an atomic model of the ADF/cofilin-F-actin complex, we have identified residues in ADF/cofilin that are essential for filament binding. Here, we have characterized the C-terminal tail of UNC-60B (a nematode ADF/cofilin isoform) as a novel determinant for its association with F-actin. Removal of the C-terminal isoleucine (Ile152) by carboxypeptidase A or truncation by mutagenesis eliminated F-actin binding activity but strongly enhanced actin depolymerizing activity. Replacement of Ile152 by Ala had a similar but less marked effect; F-actin binding was weakened and depolymerizing activity slightly enhanced. Truncation of both Arg151 and Ile152 or replacement of Arg151 with Ala also abolished F-actin binding and enhanced depolymerizing activity. Loss of F-actin binding in these mutants was accompanied by loss or greatly decreased severing activity. All of the variants of UNC-60B interacted with G-actin in an indistinguishable manner from wild type. Cryoelectron microscopy showed that UNC-60B changed the twist of F-actin to a similar extent to vertebrate ADF/cofilins. Helical reconstruction and structural modeling of UNC-60B-F-actin complex reveal how the C terminus of UNC-60B might be involved in one of the two actin-binding sites.

Muscular dystrophy: the worm turns to genetic disease.

A new animal model for studying muscular dystrophy, a mutant form of the nematode Caenorhabditis elegans, brings the power of worm genetics to bear on the search for a cure for this disease; work on this worm has already led to the identification of a novel component that can suppress the mutant phenotype.

UNC-60B, an ADF/cofilin family protein, is required for proper assembly of actin into myofibrils in Caenorhabditis elegans body wall muscle.

The Caenorhabditis elegans unc-60 gene encodes two functionally distinct isoforms of ADF/cofilin that are implicated in myofibril assembly. Here, we show that one of the gene products, UNC-60B, is specifically required for proper assembly of actin into myofibrils. We found that all homozygous viable unc-60 mutations resided in the unc-60B coding region, indicating that UNC-60B is responsible for the Unc-60 phenotype. Wild-type UNC-60B had F-actin binding, partial actin depolymerizing, and weak F-actin severing activities in vitro. However, mutations in UNC-60B caused various alterations in these activities. Three missense mutations resulted in weaker F-actin binding and actin depolymerizing activities and complete loss of severing activity. The r398 mutation truncated three residues from the COOH terminus and resulted in the loss of severing activity and greater actin depolymerizing activity. The s1307 mutation in a putative actin-binding helix caused greater activity in actin-depolymerizing and severing. Using a specific antibody for UNC-60B, we found varying protein levels of UNC-60B in mutant animals, and that UNC-60B was expressed in embryonic muscles. Regardless of these various molecular phenotypes, actin was not properly assembled into embryonic myofibrils in all unc-60 mutants to similar extents. We conclude that precise control of actin filament dynamics by UNC-60B is required for proper integration of actin into myofibrils.

Two Caenorhabditis elegans actin depolymerizing factor/cofilin proteins, encoded by the unc-60 gene, differentially regulate actin filament dynamics.

The Caenorhabditis elegans unc-60 gene encodes two actin depolymerizing factor/cofilin proteins which are implicated in the regulation of actin filament assembly in body wall muscle. We examined the interaction of recombinant UNC-60A and B proteins with actin and found that they differentially regulate actin filament dynamics. Co-pelleting assays with F-actin showed that UNC-60A depolymerized but did not remain bound to F-actin, whereas UNC-60B bound to but did not depolymerize F-actin. In the pH range of 6.8-8.0, the apparent activities of UNC-60A and B did not change although UNC-60A showed greater actin-depolymerizing activity at higher pH. These activities were further confirmed by a light scattering assay and electron microscopy. The effects of these proteins on actin polymerization were quite different. UNC-60A inhibited polymerization in a concentration-dependent manner. On the other hand, UNC-60B strongly inhibited the nucleation process but accelerated the following elongation step. However, an excess amount of UNC-60B increased the amount of unpolymerized actin. These results indicate that UNC-60A depolymerizes actin filaments and inhibits actin polymerization, whereas UNC-60B strongly binds to F-actin without depolymerizing it and, through binding to G-actin, changes the rate of actin polymerization depending on the UNC-60B:actin ratio. These data suggest that the two UNC-60 isoforms play differential roles in regulating actin filament dynamics in vivo.

Structure and stability of an immunoglobulin superfamily domain from twitchin, a muscle protein of the nematode Caenorhabditis elegans.

The NMR solution structure of an immunoglobulin superfamily module of twitchin (Ig 18') has been determined and the kinetic and equilibrium folding behaviour characterised. Thirty molecular coordinates were calculated using a hybrid distance geometry-simulated annealing protocol based on 1207 distance and 48 dihedral restraints. The atomic rms distributions about the mean coordinate for the ensemble of structures is 0.55( +/- 0.09) A for backbone atoms and 1.10( +/- 0.08) A for all heavy atoms. The protein has a topology very similar to that of telokin and the titin Ig domains and thus it falls into the I set of the immunoglobulin superfamily. The close agreement between the predicted and observed structures of Ig 18' demonstrates clearly that the I set profile can be applied in the structure prediction of immunoglobulin-like domains of diverse modular proteins. Folding studies reveal that the protein has relatively low thermodynamic stability, deltaG(H2O)U-F = 4.0 kcal mol(-1) at physiological pH. Unfolding studies suggest that the protein has considerable kinetic stability, the half life of the unfolding is greater than 40 minutes in the absence of denaturant.

Giant protein kinases: domain interactions and structural basis of autoregulation.

The myosin-associated giant protein kinases twitchin and titin are composed predominantly of fibronectin- and immunoglobulin-like modules. We report the crystal structures of two autoinhibited twitchin kinase fragments, one from Aplysia and a larger fragment from Caenorhabditis elegans containing an additional C-terminal immunoglobulin-like domain. The structure of the longer fragment shows that the immunoglobulin domain contacts the protein kinase domain on the opposite side from the catalytic cleft, laterally exposing potential myosin binding residues. Together, the structures reveal the cooperative interactions between the autoregulatory region and the residues from the catalytic domain involved in protein substrate binding, ATP binding, catalysis and the activation loop, and explain the differences between the observed autoinhibitory mechanism and the one found in the structure of calmodulin-dependent kinase I.

Substrate specificity and inhibitor sensitivity of Ca2+/S100-dependent twitchin kinases.

Myosin-associated giant protein kinases of the titin/witchin-like superfamily have previously been implicated in the regulation of muscle function, based on genetic and physiological studies. We find that recombinant constitutively active Caenorhabditis elegans and Aplysia twitchin kinase fragments differ in their catalytic activities and peptide-substrate specificities, as well as in their sensitivities to the naphthalene sulfonamide inhibitors 1-(5-chloronaphthalenesulfonyl)-1H-hexahydro-1,4-diazepine (ML-7) and 1-(5-iodonaphthalenesulfonyl)-1H-hexahydro-1,4-diazepine (ML-9). The constitutively active Aplysia twitchin kinase fragment has a remarkably high activity (Vmax > 100 mumol.min-1.mg-1) towards some substrate peptides. The autoinhibited forms of these twitchin kinases can be activated in a Ca(2+)-dependent manner by the dimeric form of the S100A1 protein (S100A1(2)). The twitchin kinase S100A1(2)-binding site can also bind Ca2+/calmodulin but neither kinase is activated by calmodulin. The data provide a functional basis for the ongoing crystallographic study of twitchin kinase fragments.

Rapid detection of mycobacteria in inflammatory necrotizing granulomas from formalin-fixed, paraffin-embedded tissue by PCR in clinically high-risk patients with acid-fast stain and culture-negative tissue biopsies.

A collection of inflammatory necrotizing granulomas (INGs) negative by acid-fast stain and culture (AFSC) were analyzed by polymerase chain reaction (PCR) for the presence of mycobacteria. Forty-two paraffin-embedded specimens with INGs were collected from patients at high risk for contracting tuberculosis. Twenty biopsies were positive and 22 were negative for mycobacteria by AFSC. Two universal primers specific for all mycobacteria were used to detected a 414 base pair (bp) fragment of 16S rRNA gene. Twenty of 20 biopsies were positive for mycobacteria by both AFSC and PCR (100%), whereas 19 of 22 biopsies negative by AFSC were positive by PCR (86%). Follow-up of patients who were PCR positive but AFSC negative identified nine patients who had subsequent biopsies. Specimens from eight of these nine patients eventually grew Mycobacterium tuberculosis. Our results demonstrate that the detection of mycobacterial DNA by this method should be used in conjunction with AFSC for the initial diagnosis of mycobacterial infection.

Ca2+/S100 regulation of giant protein kinases.

Protein phosphorylation by protein kinases plays a central regulatory role in cellular processes and these kinases are themselves tightly regulated. One common mechanism of regulation involves Ca2+-binding proteins (CaBP) such as calmodulin (CaM). Here we report a Ca2+-effector mechanism for protein kinase activation by demonstrating the specific and >1,000-fold activation of the myosin-associated giant protein kinase twitchin by Ca2+/S100A1(2). S100A1(2) is a member of a large CaBP family that is implicated in various cellular processes, including cell growth, differentiation and motility, but whose molecular actions are largely unknown. The S100A1(2)-binding site is a part of the autoregulatory sequence positioned in the active site that is responsible for intrasteric autoinhibition of twitchin kinase; the mechanism of autoinhibition based on the crystal structures of two twitchin kinase fragments is described elsewhere. Ca2+/S100 represents a likely physiological activator for the entire family of giant protein kinases involved in muscle contractions and cytoskeletal structure.

The Caenorhabditis elegans gene unc-89, required fpr muscle M-line assembly, encodes a giant modular protein composed of Ig and signal transduction domains.

Mutations in the Caenorhabditis elegans gene unc-89 result in nematodes having disorganized muscle structure in which thick filaments are not organized into A-bands, and there are no M-lines. Beginning with a partial cDNA from the C. elegans sequencing project, we have cloned and sequenced the unc-89 gene. An unc-89 allele, st515, was found to contain an 84-bp deletion and a 10-bp duplication, resulting in an in-frame stop codon within predicted unc-89 coding sequence. Analysis of the complete coding sequence for unc-89 predicts a novel 6,632 amino acid polypeptide consisting of sequence motifs which have been implicated in protein-protein interactions. UNC-89 begins with 67 residues of unique sequences, SH3, dbl/CDC24, and PH domains, 7 immunoglobulins (Ig) domains, a putative KSP-containing multiphosphorylation domain, and ends with 46 Ig domains. A polyclonal antiserum raised to a portion of unc-89 encoded sequence reacts to a twitchin-sized polypeptide from wild type, but truncated polypeptides from st515 and from the amber allele e2338. By immunofluorescent microscopy, this antiserum localizes to the middle of A-bands, consistent with UNC-89 being a structural component of the M-line. Previous studies indicate that myofilament lattice assembly begins with positional cues laid down in the basement membrane and muscle cell membrane. We propose that the intracellular protein UNC-89 responds to these signals, localizes, and then participates in assembling an M-line.

Both synchronous and asynchronous muscle isoforms of projectin (the Drosophila bent locus product) contain functional kinase domains.

In Drosophila, the large muscle protein, projectin, has very different localizations in synchronous and asynchronous muscles, suggesting that projectin has different functions in different muscle types. The multiple projectin isoforms are encoded by a single gene; however they differ significantly in size (as detected by gel mobility) and show differences in some peptide fragments, presumably indicating alternative splicing or termination. We now report additional sequence of the projectin gene, showing a kinase domain and flanking regions highly similar to equivalent regions of twitchin, including a possible autoinhibitory region. In spite of apparent differences in function, all isoforms of projectin have the kinase domain and all are capable of autophosphorylation in vitro. The projectin gene is in polytene region 102C/D where the bentD phenotype maps. The recessive lethality of bentD is associated with a breakpoint that removes sequence of the projectin kinase domain. We find that different alleles of the highly mutable recessive lethal complementation group, l(4)2, also have defects in different parts of the projectin sequence, both NH2-terminal and COOH-terminal to the bentD breakpoint. These alleles are therefore renamed as alleles of the bent locus. Adults heterozygous for projectin mutations show little, if any, effect of one defective gene copy, but homozygosity for any of the defects is lethal. The times of death can vary with allele. Some alleles kill the embryos, others are larval lethal. These molecular studies begin to explain why genetic studies suggested that l(4)2 was a complex (or pseudoallelic) locus.

Protein kinase domain of twitchin has protein kinase activity and an autoinhibitory region.

Twitchin is a 753-kDa polypeptide located in the muscle A-bands of the nematode, Caenorhabditis elegans. It consists of multiple copies of both fibronectin III and immunoglobulin C2 domains and, near the C terminus, a protein kinase domain with greatest homology to the catalytic domains of myosin light chain kinases. We have expressed and purified from Escherichia coli twitchin's protein kinase catalytic core and flanking sequences that do not include fibronectin III and immunoglobulin C2 domains. The protein was shown to phosphorylate a model substrate and to undergo autophosphorylation. The autophosphorylation occurs at a slow rate, attaining a maximum at 3 h with a stoichiometry of about 1.0 mol of phosphate/mol of protein, probably through an intramolecular mechanism. Sequence analysis of proteolytically derived phosphopeptides revealed that autophosphorylation occurred N-terminal to the catalytic core, predominantly at Thr-5910, with possible minor sites at Ser5912 and/or Ser-5913. This portion of twitchin (residues 5890-6268) was also phosphorylated in vitro by protein kinase C in the absence of calcium and phosphotidylserine, but not by cAMP-dependent protein kinase. By comparing the activities of three twitchin segments, the enzyme appears to be inhibited by the 60-amino acid residues lying just C-terminal to the kinase catalytic core. Thus, like a number of other protein kinases including myosin light chain kinases, the twitchin kinase appears to be autoregulated.

Insights into autoregulation from the crystal structure of twitchin kinase.

Many protein kinases are self-regulated by an intrasteric mechanism where part of the enzyme's structure directly inhibits the active site. This inhibitory structure is called a pseudosubstrate and specific regulators are required to remove it from the active site to allow substrates access. Removal of the pseudosubstrate sequence from members of the myosin light-chain kinase subfamily, including twitchin kinase, activates them but it is not known whether the pseudosubstrate sequence binds to the active site. Native twitchin is a 753K protein (6,839 residues) located in muscle A-bands of the nematode Caenorhabditis elegans and because of its size has not been easy to study. We have determined the crystal structure, refined to 2.8 A resolution, of a recombinant fragment (residues 5,890 to 6,262) of twitchin kinase that contains the catalytic core and a 60 residue carboxy-terminal tail. The C-terminal tail extends through the active site, wedged between the small and large lobes of the structure and making extensive contacts with the catalytic core which accounts for autoinhibition and provides direct support for the intrasteric mechanism of protein kinase regulation.

Crystallization and preliminary X-ray analysis of the auto-inhibited twitchin kinase.

An auto-inhibited fragment of twitchin kinase (residues 5890 to 6262) has been crystallized by vapor diffusion techniques using polyethylene glycol 4000 as the precipitant at pH 7.25 to 7.5 at 4 degrees C. We have found that MgSO4 and glycerol were essential for large crystal growth. The crystals belong to the orthorhombic space group P2(1)2(1)2, with unit cell dimensions of a = 144.1 A, b = 168.3 A and c = 60.6 A. They are suitable for X-ray analysis and diffract to a resolution of at least 2.8 A.

The use of deficiencies to determine essential gene content in the let-56-unc-22 region of Caenorhabditis elegans.

We have investigated the possibility of using the polymerase chain reaction to detect deletions of coding elements in the unc-22-let-56 interval on chromosome IV in the nematode Caenorhabditis elegans. Our analysis of approximately 13 kb of genomic sequence immediately to the left of the unc-22 gene resulted in the identification of four possible genes. Partial cDNAs have been identified for three of them. To determine whether any of these coding elements are essential for development, we required a method for the induction and selection of mutations in these elements. Our approach was to identify a set of formaldehyde and gamma radiation induced unc-22 mutations that mapped to the unc-22-let-56 region, and then employ polymerase chain reaction methodology to identify deficiencies that affected one or more of the four identified coding elements. Two small deficiencies were identified in this manner. Characterization of these deficiencies shows that there are no coding elements between unc-22 and let-56 (the nearest mutationally identified gene to the left of unc-22), which are required in development under laboratory conditions. We conclude that the polymerase chain reaction is a practical tool for the detection of deletions of coding elements identified in this region, and that characterization of such deficiencies provides a method for assessing whether or not these elements are required for development.

Additional sequence complexity in the muscle gene, unc-22, and its encoded protein, twitchin, of Caenorhabditis elegans.

Null mutations of the Caenorhabditis elegans unc-22 gene cause a pronounced body surface twitch associated with impaired movement and disruption of muscle structure. Partial sequence analysis of unc-22 has previously revealed that its encoded polypeptide, named twitchin, consists of a single protein kinase domain and multiple copies of both an immunoglobulin-like domain and a fibronectin type III-like domain. This paper reports additional DNA sequence information that has revealed the transcription start of unc-22, the N terminus of twitchin, and an explanation for the weak phenotype of a transposon insertion allele. These new data indicate that the unc-22 gene is 18 kb larger than previously reported and has a transcription unit of 38,308 bp. These data add 791 amino acids to the twitchin N terminus for a complete polypeptide size of 6,839 amino acids and a predicted molecular weight of 753,494. This new polypeptide sequence includes four additional copies of the above-mentioned immunoglobulin-like domains and also includes a glycine-rich sequence that might form a flexible hinge. The additional coding sequence reveals that the insertion of the Tc1 transposon, in the unc-22 allele, st139, should disrupt twitchin structure because it is located in an exon. However, cDNA sequencing has revealed that several cryptic splice donors and acceptors adjacent to the Tc1 insertion site are used to splice the transposon out of unc-22(st139) mRNA. One of these splicing events produces a near wild-type mRNA that deletes only six amino acids from twitchin, and this might explain the unusually mild phenotype associated with this mutation.

Genetic and molecular characterization of the Caenorhabditis elegans spermatogenesis-defective gene spe-17.

Two self-sterile mutations that define the spermatogenesis-defective gene spe-17 have been analyzed. These mutations affect unc-22 and fail to complement each other for both Unc-22 and spermatogenesis defects. Both of these mutations are deficiencies (hcDf1 and hDf13) that affect more than one transcription unit. Genomic DNA adjacent to and including the region deleted by the smaller deficiency (hcDf1) has been sequenced and four mRNAs (including unc-22) have been localized to this sequenced region. The three non unc-22 mRNAs are shown to be sex-specific: a 1.2-kb mRNA that can be detected in sperm-free hermaphrodites and 1.2- and 0.56-kb mRNAs found in males. hDf13 deletes at least 55 kb of chromosome IV, including all of unc-22, both male-specific mRNAs and at least part of the female-specific mRNA. hcDf1, which is approximately 15.6 kb, deletes only the 5' end of unc-22 and the gene that encodes the 0.56-kb male-specific mRNA. The common defect that apparently accounts for the defective sperm in hcDf1 and hDf13 homozygotes is deletion of the spe-17 gene, which encodes the 0.56-kb mRNA. Strains carrying two copies of either deletion are self-fertile when they are transgenic for any of four extrachromosomal array that include spe-17. We have sequenced two spe-17 cDNAs, and the deduced 142 amino acid protein sequence is highly charged and rich in serine and threonine, but shows no significant homology to any previously determined protein sequence.