Heierhorst J et al. (1994) J Biol Chem "Autophosphorylation of molluscan twitchin and interaction of its kinase domain with calcium/calmodulin."

Vilim FS, Probst WC, Heierhorst J, Weiss KR, Buku A

[J Biol Chem, 1994]

An approximately 750-kDa member of the family of giant titin/twitchin-like myosin-associated proteins was highly purified from muscle of the marine mollusc Aplysia californica. Purified twitchin was able to autophosphorylate on threonine, which demonstrates its protein serine/threonine kinase activity. cDNA sequence analysis of the cloned kinase domain of molluscan twitchin revealed that it is most closely related with the kinase domains of Caenorhabditis elegans twitchin (62-134223489dentity) and vertebrate myosin light chain kinases (45average identity). Analysis of the cDNA sequence further suggested the presence of a potential calmodulin-binding site in a putative autoinhibitory region. The functional activity of this site was demonstrated by the calcium-dependent binding of purified twitchin to immobilized calmodulin and the fact that this interaction could be competed with synthetic peptides deduced from the cDNA sequence. Furthermore, biotinylated calmodulin bound to immobilized twitchin in gel-overlay assays with nanomolar affinity (EC50 approximately equal to 70 nM). The potential regulation of twitchin by calcium/calmodulin indicates that titin-like molecules may serve dynamic functions during contraction-relaxation cycles in muscle in addition to their functions as cytoskeletal proteins.

Heierhorst J et al. (1996) Eur J Biochem "Substrate specificity and inhibitor sensitivity of Ca2+/S100-dependent twitchin kinases."

Heierhorst J, Probst WC, Tang XX, Weiss KR, Benian GM, Kemp BE, Lei JY

[Eur J Biochem, 1996]

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.

McGhee JD (1987) Biochemistry "Purification and characterization of a carboxylesterase from the intestine of the nematode Caenorhabditis elegans."

McGhee JD

[Biochemistry, 1987]

The major intestinal esterase from the nematode Caenorhabditis elegans has been purified to essential homogeneity. Starting from whole worms, the overall purification is 9000-fold with a 10% recovery of activity. The esterase is a single polypeptide chain of Mr 60,000 and is stoichiometrically inhibited by organophosphates. Substrate preferences and inhibition patterns classify the enzyme as a carboxylesterase (EC 3.1.1.1), but the physiological function is unknown. The sequence of 13 amino acid residues at the esterase N- terminus has been determined. This partial sequence shows a surprisingly high degree of similarity to the N-terminal sequence of two carboxylesterases recently isolated from Drosophila mojavensis [Pen, J., van Beeumen, J., & Beintema, J. J. (1986) Biochem. J. 238, 691-699].

Tang XX et al. (1996) West Coast Worm Meeting "CHARACTERIZATION OF A NEW TWITCHIN IN C. ELEGANS MUSCLE"

Benian GM, Fleming JT, Heierhorst J, Tang XX

[West Coast Worm Meeting, 1996]

Twitchin is a 754 kDa polypeptide encoded by the C. elegans gene unc-22 and consists of a protein kinase domain and multiple copies of FnIII and Ig domains. It is located in the A-bands of obliquely-striated body wall muscle, and functions both in muscle assembly and regulation of contraction. In unc-22 null animals, anti-twitchin staining is greatly reduced in body wall muscle, but still strong in the pharynx (Moerman et al. Genes & Develop. 2:93-105, 1988). Because unc-22 mutants do not affect pharyngeal muscle structure or function, we hypothesize that there is a pharyngeal-specific twitchin-gene. In PCR experiments designed to recover MAP-kinases from C. elegans, one clone fortuitiously encoded a peptide with highest BLAST score to twitchin. This PCR clone hybridized to YAC and cosmid clones which placed the gene on the physical map, corresponding to genetic map position -4.5 on chromosome V. Our analysis of a 5.2 kb fragment which we sequenced and the nearly complete sequence of the cosmid F17A9 from the sequencing consortium, reveals a twitchin-like protein consisting of a FnIII domain, a protein kinase domain, 8 Ig domains, a FnIII domain, and then, 8 more Ig domains. Thus, the domain organization C-terminal to the kinase differs among this new protein, twitchin and titin. The protein kinase domain of this new twitchin is approx. 52-54% identical to the kinase domains of twitchin (nematode and Aplysia), Drosophila projectin and chicken smooth muscle myosin light chain kinase. GST-fusion proteins expressing the putative kinase catalytic core (NTK1), and the catalytic core plus presumed autoinhibitory domain and Ig domain (NTK4), were tested for protein kinase activity against a myosin light chain peptide previously shown to be phosphorylated by twitchin kinase. Preliminary results show that the NTK1 Vmax was comparable to the Vmax of constitutively active twitchin kinase. Comparison of activities towards a battery of myosin light chain related peptides indicates greater similarity of new twitchin to nematode and Aplysia twitchins than to MLCK. By RT-PCR, we have detected a splice variant of new twitchin (10-20% of the molecules) which adds 15 amino acids to the beginning of the putative 60 amino acid autoinhibitory sequence. Protein kinase assays of both isoforms expressed as fusion proteins suggest that these 15 residues activate the kinase. This seems to represent a novel mechanism of protein kinase regulation. We have developed a polyclonal antiserum to a portion of new twitchin. Our preliminary results indicate reaction to a polypeptide even larger than twitchin on Western blots and immunofluorescent staining localized to the pharyngeal muscles.

Murakami S et al. (1999) Curr Biol "Life extension and stress resistance in Caenorhabditis elegans modulated by the tkr-1 gene."

Johnson TE, Murakami S

[Curr Biol, 1999]

In this Brief Communication, which appeared in the 14 September 1998 issue of Current Biology, the UV dose was reported erroneously. The dose reported was 20 J/m2 but the actual dose used was 0.4 J/cm2. Also, the gene formally referred to as tkr-1 has since been renamed old-1 (overexpression longevity determination).

Ramos CG et al. (2014) J Bacteriol "Retraction for Ramos et al., MtvR is a global small noncoding regulatory RNA in Burkholderia cenocepacia."

Feliciano JR, da Costa PJ, Ramos CG, Grilo AM, Leitao JH

[J Bacteriol, 2014]

Volume 195, no. 16, p. 35143523, 2013. A number of problems related to images published in this paper have been brought to our attention. Figure 1D contains duplicated images in lanes S and LE, and Fig. 4D and 6B contain images previously published in articles in this journal and in Microbiology and Microbial Pathogenesis, i.e., the following: C. G. Ramos, S. A. Sousa, A. M. Grilo, J. R. Feliciano, and J. H. Leitao, J. Bacteriol. 193:15151526, 2011. doi:10.1128/JB.01374-11. S. A. Sousa, C. G. Ramos, L. M. Moreira, and J. H. Leitao, Microbiology 156:896908, 2010. doi:10.1099/mic.0.035139-0. C. G. Ramos, S. A. Sousa, A. M. Grilo, L. Eberl, and J. H. Leitao, Microb. Pathog. 48:168177, 2010. doi: 10.1016/j.micpath.2010.02.006. Therefore, we retract the paper. We deeply regret this situation and apologize for any inconvenience to the editors and readers of Journal of Bacteriology, Microbial Pathogenesis, and Microbiology.

Nillegoda NB et al. (2015) Nature "Crucial HSP70 co-chaperone complex unlocks metazoan protein disaggregation."

Berynskyy M, Morimoto RI, Bukau B, Stengel F, Kirstein J, Szlachcic A, Arnsburg K, Stank A, Scior A, Nillegoda NB, Gao X, Guilbride DL, Aebersold R, Wade RC, Mayer MP

[Nature, 2015]

Protein aggregates are the hallmark of stressed and ageing cells, and characterize several pathophysiological states. Healthy metazoan cells effectively eliminate intracellular protein aggregates, indicating that efficient disaggregation and/or degradation mechanisms exist. However, metazoans lack the key heat-shock protein disaggregase HSP100 of non-metazoan HSP70-dependent protein disaggregation systems, and the human HSP70 system alone, even with the crucial HSP110 nucleotide exchange factor, has poor disaggregation activity in vitro. This unresolved conundrum is central to protein quality control biology. Here we show that synergic cooperation between complexed J-protein co-chaperones of classes A and B unleashes highly efficient protein disaggregation activity in human and nematode HSP70 systems. Metazoan mixed-class J-protein complexes are transient, involve complementary charged regions conserved in the J-domains and carboxy-terminal domains of each J-protein class, and are flexible with respect to subunit composition. Complex formation allows J-proteins to initiate transient higher order chaperone structures involving HSP70 and interacting nucleotide exchange factors. A network of cooperative class A and B J-protein interactions therefore provides the metazoan HSP70 machinery with powerful, flexible, and finely regulatable disaggregase activity and a further level of regulation crucial for cellular protein quality control.

Tang XX et al. (1997) International C. elegans Meeting "INTESTINAL BRUSH BORDER TWITCHIN"

Benian GM, Fleming JT, Heierhorst J, Tang XX

[International C. elegans Meeting, 1997]

Previously we noted that a new twitchin is encoded by cosmids F17A9 and F12F3 on chromosome V. Sequence obtained from the Sequencing Project was analyzed by GeneMark and our sequencing of RT-PCR products and cDNA clones. We can account for coding sequence which specifies a 7,030 amino acid polypeptide distributed over a 36,983 bp gene interrupted by 29 introns. The tenth intron (9,497 bp) contains the coding sequence for the mutationally-defined exp-2 potassium channel (L. Avery) on the opposite strand. New twitchin consists of 33 Ig, 7 FnIII domains, and a protein kinase domain. It also has a 1426 residue region containing 8 copies of a ~30 residue motif. Bacterially expressed kinase catalytic core has protein kinase activity towards peptides derived from myosin light chains, with a Vmax similar to the Vmax of constitutively active twitchin kinase. The 60 amino acids lying just C-terminal to the kinase domain partially inhibit kinase activity. Adding 15 residues to the beginning of these 60 residues, by alternative splicing, relieves much of this inhibition. A polyclonal antiserum (EU48) reacts to a twitchin-sized polypeptide on western blots and localizes to the intestinal brush border by immunofluorescent staining. In fact, EU48 co-localizes with monoclonal MH33 (R. Francis) which was shown previously to be restricted to the terminal web. In vertebrates, the terminal web is a region where the microvillar actin bundles are anchored in a complex network of filaments containing myosin II, spectrins, tropomyosin, microtubules, intermediate filaments, and a molecule similar to titin (a twitchin homolog). Several lethals with death at early larval stages have been mapped by Avery et al. to either side of exp-2 (-0.32 mu), the closest being let-402 (-0.34 mu). After determining the 5' end of the gene, we will try to rescue these lethals with new twitchin.

Miller DM et al. (1992) Worm Breeder's Gazette "unc-4 LacZ Expression in A-Type Motor Neurons"

Miller DM, Niemeyer CJ

[Worm Breeder's Gazette, 1992]

unc-4 LacZ expression in A-type motor neurons David M. Miller and Charles J. Niemeyer, Dept. of Cell Biology, Duke Univ. Medical Ctr, Durham, NC 27710

Sommer RJ et al. (1994) Worm Breeder's Gazette "Evolution of Vulva-Formation: Part II: Species With a Central Vulva"

Sommer RJ, Sternberg PW

[Worm Breeder's Gazette, 1994]

Evolution of vulva-formation: Part II: Species with a central vulva Ralf J. Sommer & Paul W. Sternberg, California Institute of Technology, Division of Biology 156-29, Pasadena, CA 91125