Secondary EWSR1 gene abnormalities in SMARCB1-deficient tumors with 22q11-12 regional deletions: Potential pitfalls in interpreting EWSR1 FISH results.

SMARCB1 inactivation occurs in a variety of tumors, being caused by various genetic mechanisms. Since SMARCB1 and EWSR1 genes are located close to each other on chromosome 22, larger SMARCB1 deletions may encompass the EWSR1 locus. Herein, we report four cases with SMARCB1-deletions showing concurrent EWSR1 gene abnormalities by FISH, which lead initially to misinterpretations as EWSR1-rearranged tumors. Our study group included various morphologies: a poorly differentiated chordoma, an extrarenal rhabdoid tumor, a myoepithelial carcinoma, and a proximal-type epithelioid sarcoma. All cases showed loss of SMARCB1 (INI1) by immunohistochemistry (IHC) and displayed characteristic histologic features for the diagnoses. The SMARCB1 FISH revealed homozygous or heterozygous deletions in three and one case, respectively. The co-hybridized EWSR1 probes demonstrated either unbalanced split signals or heterozygous deletion in two cases each. The former suggested bona fide rearrangement, while the latter resembled an unbalanced translocation. However, all the FISH patterns were quite complex and distinct from the simple and uniform split signals seen in typical EWSR1 rearrangements. We conclude that in the context of 22q11-12 regional alterations present in SMARCB1-deleted tumors, simultaneous EWSR1 involvement may be misinterpreted as equivalent to EWSR1 rearrangement. A detailed clinicopathologic correlation and supplementing the EWSR1 FISH assay with complementary methodology is mandatory for correct diagnosis. © 2016 Wiley Periodicals, Inc.

Purification of F plasmid-encoded native TraC from Escherichia coli by affinity chromatography on calmodulin Sepharose.

We have enriched several native bacterial proteins from Escherichia coli by chromatography on the immobilized eukaryotic Ca(2+)-binding protein, calmodulin. These bacterial proteins bound in a Ca(2+)-dependent manner to calmodulin, and were released by the addition of the Ca(2+)-chelator, EGTA, similar to many eukaryotic calmodulin-binding proteins. One of the bacterial proteins, F factor-encoded TraC, was purified to apparent homogeneity by an additional chromatographic step, anion exchange chromatography on MonoQ. Experiments with four chemically distinct calmodulin antagonists (R24571, Compound 48/80, melittin, and W7) showed that all of these substances inhibited the binding of purified TraC to calmodulin at effective concentrations comparable to those required for inhibiting in vitro binding of eukaryotic calmodulin-binding proteins. Three further bacterial proteins were identified as calmodulin-binding proteins: SecA, GlpD, and GlpC. We suggest that also these native bacterial proteins might be isolated by the unusual purification procedure including affinity chromatography on calmodulin Sepharose. Whether the identified proteins bind to, and are regulated by, putative bacterial calmodulin-like proteins in Escherichia coli remains to be established.

Bioorthogonal Chemoenzymatic Functionalization of Calmodulin for Bioconjugation Applications.

Calmodulin (CaM) is a widely studied Ca(2+)-binding protein that is highly conserved across species and involved in many biological processes, including vesicle release, cell proliferation, and apoptosis. To facilitate biophysical studies of CaM, researchers have tagged and mutated CaM at various sites, enabling its conjugation to fluorophores, microarrays, and other reactive partners. However, previous attempts to add a reactive label to CaM for downstream studies have generally employed nonselective labeling methods or resulted in diminished CaM function. Here we report the first engineered CaM protein that undergoes site-specific and bioorthogonal labeling while retaining wild-type activity levels. By employing a chemoenzymatic labeling approach, we achieved selective and quantitative labeling of the engineered CaM protein with an N-terminal 12-azidododecanoic acid tag; notably, addition of the tag did not interfere with the ability of CaM to bind Ca(2+) or a partner protein. The specificity of our chemoenzymatic labeling approach also allowed for selective conjugation of CaM to reactive partners in bacterial cell lysates, without intermediate purification of the engineered protein. Additionally, we prepared CaM-affinity resins that were highly effective in purifying a representative CaM-binding protein, demonstrating that the engineered CaM remains active even after surface capture. Beyond studies of CaM and CaM-binding proteins, the protein engineering and surface capture methods described here should be translatable to other proteins and other bioconjugation applications.

RNA sequencing identifies fusion of the EWSR1 and YY1 genes in mesothelioma with t(14;22)(q32;q12).

Mesothelioma is a rare but very aggressive tumor derived from mesothelial cells. A number of often complex but nonrandom cytogenetic abnormalities have been found in these tumors, resulting in loss of chromosome bands 14q32 and 22q12 in more than 35% of the cases. In this study, we used RNA sequencing to search for fusion transcripts in a mesothelioma carrying a t(14;22)(q32;q12) as the sole chromosomal aberration and found an EWSR1-YY1 and its reciprocal YY1-EWSR1 fusion transcript. Screening 15 additional cases of mesothelioma from which we had RNA but no cytogenetic information, we identified one more tumor carrying an EWSR1-YY1 fusion gene but not the reciprocal YY1-EWSR1 transcript. RT-polymerase chain reaction and sequencing showed that in both cases exon 8 of EWSR1 (nucleotide 1,139, accession number NM_013986 version 3, former exon 7 in sequence with accession number X66899) was fused to exon 2 of YY1 (nucleotide 1,160, accession number NM_003403 version 3). The EWSR1 breakpoint in exon 8 in the EWSR1-YY1 chimeric transcript is similar to what is found in other fusions involving EWSR1 such as EWSR1-FLI1, EWSR1-DDIT3, and EWSR1-ATF1. The EWSR1-YY1-encoded protein is an abnormal transcription factor with the transactivation domain of EWSR1 and the DNA-binding domain of YY1. This is the first study to detect a specific fusion gene in mesothelioma (the reason how frequent the EWSR1-YY1 fusion is remains uncertain) and also the first time that direct involvement of YY1 in oncogenesis has been demonstrated.

The primary identification of a calcineurin A subunit-like protein in plants.

Many types of serine/threonine protein phosphatase have been cloned and characterized in plants, such as Type-1 serine/threonine protein phosphatase (PP1), Type-2A serine/threonine protein phosphatase (PP2A), Type-2C serine/threonine protein phosphatase (PP2C). However no Type-2B serine/threonine protein phosphatase (PP2B, calcineurin), or calcineurin A subunit-like protein (CaNAL), has been identified. We detected protein phosphatase activity in mixtures of CaM-binding proteins from three plants (Nicotiana tabacum, Brassica oleracea and Arabidopsis thaliana). Two-dimensional electrophoresis (2-D) and Western blot analysis with an anti-rat CNA antibody revealed a small protein of 60 kDa that we believe is a CaNAL. The isoelectric point (pI) of this protein in N. tabacum was approximately 5.69. The protein phosphatase activity in the mixture of CaM-binding proteins from N. tabacum was regulated by Ca(2+) and Calmodulin (CaM) with either RII peptides or pNPP as substrate. The immunosuppressive drugs, CsA and FK506, also inhibited the protein phosphatase activity significantly.

In vitro characterization of native mammalian smooth-muscle protein synaptopodin 2.

An analysis of the primary structure of the actin-binding protein fesselin revealed it to be the avian homologue of mammalian synaptopodin 2 [Schroeter, Beall, Heid, and Chalovich (2008) Biochem. Biophys. Res. Commun. 371, 582-586]. We isolated two synaptopodin 2 isoforms from rabbit stomach that corresponded to known types of human synaptopodin 2. The purification scheme used was that developed for avian fesselin. These synaptopodin 2 forms shared several key functions with fesselin. Both avian fesselin and mammalian synaptopodin 2 bound to Ca(2+)-calmodulin, alpha-actinin and smooth-muscle myosin. In addition, both proteins stimulated the polymerization of actin in a Ca(2+)-calmodulin-dependent manner. Synaptopodin 2 has never before been shown to polymerize actin in the absence of alpha-actinin, to polymerize actin in a Ca(2+)-calmodulin-dependent manner, or to bind to Ca(2+)-calmodulin or myosin. These properties are consistent with the proposed function of synaptopodin 2 in organizing the cytoskeleton.

Ca(2+)/Calmodulin-binding proteins from the C. elegans proteome.

Calmodulin (CaM) is the primary Ca(2+)-sensor that regulates a wide variety of cellular processes in eukaryotes. Although many Ca(2+)/CaM-binding proteins have been identified, very few such proteins could be found from the genome-wide protein-protein interaction maps of Caenorhabditis elegans constructed by yeast two-hybrid screening. Using a genotype-phenotype conjugation method called mRNA-display, we performed a selection for Ca(2+)/CaM-binding proteins from a proteome library of C. elegans. The method allowed the identification of 9 known and 47 previously uncharacterized Ca(2+)-dependent CaM-binding proteins from the adult worm proteome. The Ca(2+)/CaM-binding properties of these proteins were characterized and their binding motifs were identified. The availability of such information could facilitate our understanding of the signaling pathways mediated by Ca(2+)/CaM in C. elegans. Due to its simplicity and efficiency, the method could be readily applied to examine the Ca(2+)-dependent binding partners of numerous other Ca(2+)-binding proteins, which may play important roles in many signaling pathways in C. elegans.

[The influence of caldesmon on strong binding of myosin with actin in denervated rat skeletal muscles].

The effect of caldesmon (CaD) on conformational changes in F-actin modified by fluorescent probe TRITC-phalloidin was investigated by polarized fluorimetry. Changes were induced by a subfragment-1 (S-1) of myosin in the absence or presence of CaD in ghost muscle fibers obtained from intact and denervated slow (SOL) and fast (EDL) skeletal muscles of rats. S-1 binding to actin of both SOL and EDL muscles was shown to cause changes in polarized parameters of TRITC-phalloidin typical for a strong actin-myosin binding as well as of transition ofactin subunits from "off" to "on" state. CaD inhibits this significantly. Denervation atrophy inhibits the effect of S-1 as well but does not affect the capability of CaD decreasing the formation of strong binding in actomyosin complex. It is supposed that CaD "freezes" F-actin structure in "off" state. The denervation atrophy has no effect on CaD responsibility to bind thin filaments and to switch "off" actin monomers.

Isolation, characterization, and bioinformatic analysis of calmodulin-binding protein cmbB reveals a novel tandem IP22 repeat common to many Dictyostelium and Mimivirus proteins.

A novel calmodulin-binding protein cmbB from Dictyostelium discoideum is encoded in a single gene. Northern analysis reveals two cmbB transcripts first detectable at 4 h during multicellular development. Western blotting detects an approximately 46.6 kDa protein. Sequence analysis and calmodulin-agarose binding studies identified a "classic" calcium-dependent calmodulin-binding domain (179IPKSLRSLFLGKGYNQPLEF198) but structural analyses suggest binding may not involve classic alpha-helical calmodulin-binding. The cmbB protein is comprised of tandem repeats of a newly identified IP22 motif ([I,L]Pxxhxxhxhxxxhxxxhxxxx; where h = any hydrophobic amino acid) that is highly conserved and a more precise representation of the FNIP repeat. At least eight Acanthamoeba polyphaga Mimivirus proteins and over 100 Dictyostelium proteins contain tandem arrays of the IP22 motif and its variants. cmbB also shares structural homology to YopM, from the plague bacterium Yersenia pestis.

The transcriptional coactivator CAMTA2 stimulates cardiac growth by opposing class II histone deacetylases.

Postnatal cardiac myocytes respond to diverse signals by hypertrophic growth and activation of a fetal gene program. In an effort to discover regulators of cardiac hypertrophy, we performed a eukaryotic expression screen for activators of the atrial natriuretic factor (ANF) gene, a cardiac-specific marker of hypertrophic signaling. We discovered that a family of transcriptional coactivators, called CAMTAs, promotes cardiomyocyte hypertrophy and activates the ANF gene, at least in part, by associating with the cardiac homeodomain protein Nkx2-5. The transcriptional activity of CAMTAs is governed by association with class II histone deacetylases (HDACs), which negatively regulate cardiac growth. Mice homozygous for a mutation in a CAMTA gene are defective in cardiac growth in response to pressure overload and neurohumoral signaling, whereas mice lacking HDAC5, a class II HDAC, are sensitized to the prohypertrophic actions of CAMTA. These findings reveal a transcriptional regulatory mechanism that modulates cardiac growth and gene expression by linking hypertrophic signals to the cardiac genome.

Isolation of a calmodulin-binding transcription factor from rice (Oryza sativa L.).

Calmodulin (CaM) regulates diverse cellular functions by modulating the activities of a variety of enzymes and proteins. However, direct modulation of transcription factors by CaM has been poorly understood. In this study, we isolated a putative transcription factor by screening a rice cDNA expression library by using CaM:horse-radish peroxidase as a probe. This factor, which we have designated OsCBT (Oryza sativa CaM-binding transcription factor), has structural features similar to Arabidopsis AtSRs/AtCAMTAs and encodes a 103-kDa protein because it contains a CG-1 homology DNA-binding domain, three ankyrin repeats, a putative transcriptional activation domain, and five putative CaM-binding motifs. By using a gel overlay assay, gel mobility shift assays, and site-directed mutagenesis, we showed that OsCBT has two different types of functional CaM-binding domains, an IQ motif, and a Ca(2+)-dependent motif. To determine the DNA binding specificity of OsCBT, we employed a random binding site selection method. This analysis showed that OsCBT preferentially binds to the sequence 5'-TWCG(C/T)GTKKKKTKCG-3' (W and K represent A or C and T or G, respectively). OsCBT was able to bind this sequence and activate beta-glucuronidase reporter gene expression driven by a minimal promoter containing tandem repeats of these sequences in Arabidopsis leaf protoplasts. Green fluorescent protein fusions of two putative nuclear localization signals of OsCBT, a bipartite and a SV40 type, were predominantly localized in the nucleus. Most interestingly, the transcriptional activation mediated by OsCBT was inhibited by co-transfection with a CaM gene. Taken together, our results suggest that OsCBT is a transcription activator modulated by CaM.

Isolation and characterization of Dictyostelium thymidine kinase 1 as a calmodulin-binding protein.

Probing of a cDNA expression library from multicellular development of Dictyostelium discoideum using a recombinant radiolabelled calmodulin probe (35S-VU1-CaM) led to the isolation of a cDNA encoding a putative CaM-binding protein (CaMBP). The cDNA contained an open reading frame of 951 bp encoding a 227aa polypeptide (25.5 kDa). Sequence comparisons led to highly significant matches with cytosolic thymidine kinases (TK1; EC 2.7.1.21) from a diverse number of species including humans (7e-56; 59% Identities; 75% Positives) indicating that the encoded protein is D. discoideum TK1 (DdTK1; ThyB). DdTK1 has not been previously characterized in this organism. In keeping with its sequence similarity with DdTK1, antibodies against humanTK1 recognize DdTK1, which is expressed during growth but decreases in amount after starvation. A CaM-binding domain (CaMBD; 20GKTTELIRRIKRFNFANKKC30) was identified and wild type DdTK1 plus two constructs (DdTK deltaC36, DdTK deltaC75) possessing the domain were shown to bind CaM in vitro but only in the presence of calcium while a construct (DdTK deltaN72) lacking the region failed to bind to CaM. Thus, DdTK1 is a Ca2+-dependent CaMBP. Sequence alignments against TK1 from vertebrates to viruses show that CaM-binding region is highly conserved. The identified CaMBD overlaps the ATP-binding (P-loop) domain suggesting CaM might affect the activity of this kinase. Recombinant DdTK is enzymatically active and showed stimulation by CaM (113+/-0.5%) an in vitro enhancement that was prevented by co-addition of the CaM antagonists W7 (91.2+/-0.8%) and W13 (96.6+/-0.6%). The discovery that TK1 from D. discoideum, and possibly other species including humans and a large number of human viruses, is a Ca2+-dependent CaMBP opens up new avenues for research on this medically relevant protein.

Identification of Ca2+-dependent calmodulin-binding proteins in rat spermatogenic cells as complexes of the heat-shock proteins.

Ca2+-calmodulin (CaM)-binding proteins in rat testes were characterized by assays for CaM-binding activity using the CaM-overlay method on transblots of electrophoresed gels and purification by gel-filtration, ion exchange, and adsorption chromatographies. A major CaM-binding protein complex (CaMBP) was identified and found to be comprised of three proteins with molecular masses 110, 100, and 70 kDa. Amino acid sequence analyses of lysylendopeptidase digests from these proteins indicated that all of the constituents of CaMBP are very similar to the members of the heat-shock protein family, i.e., the 110-kDa protein is similar to the APG-2/94 kDa rat ischemia-responsive protein, the 100-kDa protein is similar to the rat counterpart of the mouse APG-1/94 kDa osmotic stress protein, and the 70-kDa protein is similar to the rat testis-specific major heat-shock protein (HSP70). Immunohistochemistry using anti-CaMBP and anti-CaM antibodies demonstrated that CaMBP was co-localized with CaM in the cytoplasm of pachytene spermatocytes and nuclei of round spermatids. In addition, CaMBP, but not CaM, was localized at a high level in the residual bodies of elongated spermatids. The possible relevance of CaMBP to regulation of cell cycle progression and spermatogenesis is discussed in this paper.

Studies of calmodulin-dependent regulation.

Methods are presented for purifying bovine testes calmodulin and the calmodulin-regulated plasma-membrane calcium ATPase from human erythrocytes by calcium dependent affinity chromatography. The assay of CaM Kinase II using a synthetic peptide substrate is also described.

Calmodulin-binding proteins in bryophytes: identification of abscisic acid-, cold-, and osmotic stress-induced genes encoding novel membrane-bound transporter-like proteins.

Plant responses to environmental stresses are mediated in part by signaling processes involving cytosolic Ca2+ and a Ca(2+)-binding protein, calmodulin. Screening with radiolabeled calmodulin of a cDNA library of the moss Physcomitrella patens resulted in identification of genes encoding novel membrane transporter-like proteins, MCamb1 and MCamb2. These proteins each had a central hydrophobic domain with two putative membrane spans and N- and C-terminal hydrophilic domains, and showed sequence similarity to mammalian inward rectifier potassium channels. Calmodulin binds to MCamb1 and MCamb2 via interaction with basic amphiphilic amino acids in the C-terminal domain. Levels of MCamb1 and MCamb2 transcripts increased dramatically following treatment with low temperature, hyperosmotic solutes, and the stress hormone abscisic acid, all of which were previously shown to increase cellular tolerance to freezing stress. These results suggest that calmodulin participates in cellular signaling events leading to enhancement of stress resistance through regulation of novel transporter-like proteins.

Evidence for the direct interaction between calmodulin and the human epidermal growth factor receptor.

Previous work from our laboratory has demonstrated that the Ca(2+)-calmodulin complex inhibits the intrinsic tyrosine kinase activity of the epidermal growth factor receptor (EGFR), and that the receptor can be isolated by Ca(2+)-dependent calmodulin-affinity chromatography [San José, Bengurija, Geller and Villalobo (1992) J. Biol. Chem. 267, 15237-15245]. Moreover, we have demonstrated that the cytosolic juxtamembrane region of the human receptor (residues 645-660) binds calmodulin in a Ca(2+)-dependent manner when this segment forms part of a recombinant fusion protein [Martijn-Nieto and Villalobo (1998) Biochemistry 37, 227-236]. However, demonstration of the direct interaction between calmodulin and the whole receptor has remained elusive. In this work, we show that calmodulin, in the presence of Ca(2+), forms part of a high-molecular-mass complex built upon covalent cross-linkage of the human EGFR immunoprecipitated from two cell lines overexpressing this receptor. Although several calmodulin-binding proteins co-immunoprecipitated with the EGFR, suggesting that they interact with the receptor, we demonstrated using overlay techniques that biotinylated calmodulin binds directly to the receptor in a Ca(2+)-dependent manner without the mediation of any adaptor calmodulin-binding protein. Calmodulin binds to the EGFR with an apparent dissociation constant (K'(d)) of approx. 0.2-0.3 microM. Treatment of cells with epidermal growth factor, or with inhibitors of protein kinase C and calmodulin-dependent protein kinase II, or treatment of the immunoprecipitated receptor with alkaline phosphatase, does not significantly affect the binding of biotinylated calmodulin to the receptor.

Phosphorylation of molluscan twitchin by the cAMP-dependent protein kinase.

Catch in certain molluscan muscles is released by an increase in cAMP, and it was suggested that the target of cAMP-dependent protein kinase (PKA) is the high molecular weight protein twitchin [Siegman, M. J., Funabara, J., Kinoshita, S., Watabe, S., Hartshorne, D. J., and Butler, T. M. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 5384-5388]. This study was carried out to investigate the phosphorylation of twitchin by PKA. Twitchin was isolated from Mytilus catch muscles and was phosphorylated by PKA to a stoichiometry of about 3 mol of P/mol of twitchin. There was no evidence of twitchin autophosphorylation. Two phosphorylated peptides were isolated and sequenced, termed D1 and D2. Additional cDNA sequence for twitchin was obtained, and the D2 site was located at the C-terminal side of the putative kinase domain in a linker region between two immunoglobulin C2 repeats. Excess PKA substrates, e.g., D1 and D2, blocked the reduction in force on addition of cAMP, confirming the role for PKA in regulating catch. Papain proteolysis of (32)P-labeled twitchin from permeabilized muscles showed that the D1 site represented about 50% of the (32)P labeling. Proteolysis of in-situ twitchin with thermolysin suggested that the D1 and D2 sites were at the N- and C-terminal ends of the molecule, respectively. Thermolysin proteolysis also indicated that D1 and D2 were major sites of phosphorylation by PKA. The direct phosphorylation of twitchin by PKA is consistent with a regulatory role for twitchin in the catch mechanism and probably involves phosphorylation at the D1 and D2 sites.

Rab27a: A key to melanosome transport in human melanocytes.

Normal pigmentation depends on the uniform distribution of melanin-containing vesicles, the melanosomes, in the epidermis. Griscelli syndrome (GS) is a rare autosomal recessive disease, characterized by an immune deficiency and a partial albinism that has been ascribed to an abnormal melanosome distribution. GS maps to 15q21 and was first associated with mutations in the myosin-V gene. However, it was demonstrated recently that GS can also be caused by a mutation in the Rab27a gene. These observations prompted us to investigate the role of Rab27a in melanosome transport. Using immunofluorescence and immunoelectron microscopy studies, we show that in normal melanocytes Rab27a colocalizes with melanosomes. In melanocytes isolated from a patient with GS, we show an abnormal melanosome distribution and a lack of Rab27a expression. Finally, reexpression of Rab27a in GS melanocytes restored melanosome transport to dendrite tips, leading to a phenotypic reversion of the diseased cells. These results identify Rab27a as a key component of vesicle transport machinery in melanocytes.

[Isolation, purification and identification of metallothionein from strain BD102 of Hansenula anomala].

Metallothionein (MTs) in Cu and Cd resistant strain BD102 of Hansenula anomala were induced by administration of Cu2+ and Cd2+. These proteins were isolated and purified by Sephadex G-50 and subsequent DEAE-Sepharose CL-6B, then Sephadex G-25 for desalation. There were two isoform MTs by Cu(Cu-MTs), one form induced by Cd (Cd-MT). The molecular weights of the Cu-MTs and the Cd-MT were about 7 kD and 7.5 kD respectively. Exposure of Hansenula anomala to copper salts stimulated formation of two isoform Cu-MTs with a cysteine content of 6.6-6.8% and had 60 amino acids. Exposure of Hansenula anomala to cadmium, stimulated formation of Cd-MT with a cystein content of 10% was synthesized and had 61 amino acids. 4 atom Cu or Cd/mole MTs.