Unusual Fusion Research Articles

Unusual fusion gene rearrangements in patients with nodular fasciitis: a study of rare and novel USP6 fusion partners with a review of the literature

AimsThis retrospective non-randomised study aims to identify new and rare fusion partners with USP6 in the setting of nodular fasciitis. It has been proven, that nodular fasciitis can harbour different...

Facilitated orthodontic tooth movement with periodontal intervention: A case report

Maxillary central incisors play a significant role in the facial aesthetics as it forms a major component of smile. We report a 27 years old male patient undergoing orthodontic treatment for the forceful traction of impacted maxillary central incisors into the occlusion who presented with a fusion of the gingiva and lips in the maxillary anterior region obliterating the maxillary labial vestibule and entrapping the orthodontic retractors earlier placed. Vestibuloplasty was carried out using scalpel and electrocautery to regain the vestibular depth followed by exposure and traction of the impacted maxillary central incisors into occlusion. The patient achieved excellent results which was realised at one-year follow-up, by which time orthodontic treatment was also completed. The case is being described because of the unusual fusion of lips and gingiva, and hence requires careful treatment planning for successful outcomes.

Exploring the structure and function of the peptidase-ABC transporter fusion protein ZmaM.

Bacteria compete with other microbes for scarce resources through bacterial warfare. One way they accomplish this is by producing and secreting antifungal, antiprotist, and antibiotic compounds. To protect themselves from these toxic compounds, some bacteria produce inactive forms of these molecules and activate them once they are exported. One class of these prodrug molecules is produced with N-acyl-D-Asn prodrug motifs that are then processed by two different kinds of prodrug-activating peptidases. Type I peptidases are serine peptidases composed of a periplasmic S12 hydrolase domain and a three-helix transmembrane domain (TMD). The most well-known type I peptidase is ClbP, which activates the E. coli genotoxin colibactin. Type II peptidases contain an additional ABC half-transporter module fused to the C-terminus of the three-helix TMD. While we are beginning to understand the structure and function of type I peptidases, little is known about type II peptidases. We are investigating the type II peptidase ZmaM, involved in synthesizing the B. cereus antimicrobial zwittermicin, as a model system for understanding the structure and function of these fused proteins. We are pursuing high-resolution structures using cryoEM to understand the overall placement of the two functional modules. Concurrently, we are also using in vitro and in vivo activity assays to evaluate the peptidase, transport and ATPase activities of ZmaM and probing whether any coordination exists between its two functional modules. Understanding the function of this unusual fusion protein gives insight into the production of zwittermicin and other bioactive compounds that use type II peptidases in their synthesis. This work can also facilitate bioengineering studies into modifying ZmaM's activity for zwittermicin production or altering its specificity for designer drugs that benefit from contiguous secretion and processing.

A novel thioredoxin glutathione reductase from evolutionary ancient metazoan Hydra

Thioredoxin (Trx) and glutathione disulfide (GSSG), are regenerated in reduced state by thioredoxin reductase (TrxR) and glutathione reductase (GR) respectively. A novel protein thioredoxin glutathione reductase (TGR) capable of reducing Trx as well as GSSG, linking two redox systems, has only been reported so far from parasitic flat worms and mammals. For the first time, we report a multifunctional antioxidant enzyme TGR from the nonparasitic, nonmammalian cnidarian Hydra vulgaris (HvTGR) which is a selenoprotein with unusual fusion of a TrxR domain with glutaredoxin (Grx) domain. We have cloned and sequenced HvTGR which encodes a polypeptide of 73 kDa. It contains conserved sequence CPYC of Grx domain, and CVNVGC and GCUG domains of thioredoxin reductase. Phylogenetic analysis revealed HvTGR to be closer to TGR from mammals rather than to TGR from parasitic helminths. We then subcloned HvTGR in plasmid pSelExpress-1 and expressed it in HEK293T cells to ensure selenocysteine incorporation. Purified HvTGR showed Grx, glutathione reductase and TrxR activities. Both thioredoxin and GSSG disulfide reductase activities were inhibited by 1-Chloro-2,4-dinitrobenzene (DNCB) supporting the existence of an essential selenocysteine residue. HvTGR expression was induced in response to H2O2 in Hydra. Interestingly, inhibition of HvTGR by DNCB, inhibited regeneration in Hydra indicating its involvement in other cellular processes.

An unreported infraspinatus muscle variation—two-headed infraspinatus minor muscle and three-headed fusion with the teres minor muscle

The infraspinatus muscle is situated under the scapular spine in the infraspinous fossa and inserts into the greater tuberosity of the humerus. It is a component of a crucial shoulder muscle group, the rotator cuff. There are a few interesting additional muscles in the infraspinal region. In the literature they are called the infraspinatus superficialis, infraspinatus minor and infraspinatus accessory muscles. The infraspinatus minor muscle is described as a superficial muscle bundle running under the scapular spine. During routine anatomical dissection, an unreported variation of the infraspinatus minor muscle was found. It derived from the inferior surface of the scapular spine and the infraspinous fossa. It had two heads. The superior head inserted on the greater tuberosity of the humerus. The inferior head inserted on the tendinous part of the infraspinatus muscle. There was also an unusual fusion of the infraspinatus muscle with the teres minor muscle. In this paper we will discuss the anatomical and physiological relationships of this morphological variation.

Doubly Fused Unsymmetrical Calixdicarbahexaphyrins.

Three novel doubly fused unsymmetrical calixdicarbahexaphyrins were synthesized by mild acid-catalyzed (4+2) condensation of dicarbatetrapyrrane with dipyrroethene diol followed by oxidation. The condensation formed doubly fused calixdicarbahexaphyrins instead of π-conjugated dicarbahexaphyrins, due to the unusual fusion of the pyrrole N with the α-carbon of the adjacent pyrrole ring to form a tripentacyclic ring and one usual fusion of the pyrrole N with the adjacent phenylene C to form a fused moiety containing two pentacycles and one hexacycle ring. Both fusions occurred on one side of the macrocycle, making the macrocycles unsymmetric. The crystal structure obtained for one of the macrocycles exhibited a saddle-shaped structure with two benzene rings and four pyrrole rings connected via two ethylene and four methene meso-carbon atoms. The crystal structure also revealed unusual fusions in the macrocyclic framework and the presence of one sp3 carbon that disrupts the π-electron delocalization. 1H, 1H-1H COSY, NOESY, 13C, and HMBC NMR techniques were used to characterize the macrocycles. The absorption spectra of the macrocycles showed one intense sharp band at ∼485 nm along with a shoulder in the lower-energy region, suggesting its non-aromatic nature. Electrochemical studies indicated their electron rich nature, and DFT/TD-DFT studies corroborated the experimental observations.

Biochemical and structural characterizations of thioredoxin reductase selenoproteins of the parasitic filarial nematodes Brugia malayi and Onchocerca volvulus

Enzymes in the thiol redox systems of microbial pathogens are promising targets for drug development. In this study we characterized the thioredoxin reductase (TrxR) selenoproteins from Brugia malayi and Onchocerca volvulus, filarial nematode parasites and causative agents of lymphatic filariasis and onchocerciasis, respectively. The two filarial enzymes showed similar turnover numbers and affinities for different thioredoxin (Trx) proteins, but with a clear preference for the autologous Trx. Human TrxR1 (hTrxR1) had a high and similar specific activity versus the human and filarial Trxs, suggesting that, in vivo, hTrxR1 could possibly be the reducing agent of parasite Trxs once they are released into the host. Both filarial TrxRs were efficiently inhibited by auranofin and by a recently described inhibitor of human TrxR1 (TRi-1), but not as efficiently by the alternative compound TRi-2. The enzyme from B. malayi was structurally characterized also in complex with NADPH and auranofin, producing the first crystallographic structure of a nematode TrxR. The protein represents an unusual fusion of a mammalian-type TrxR protein architecture with an N-terminal glutaredoxin-like (Grx) domain lacking typical Grx motifs. Unlike thioredoxin glutathione reductases (TGRs) found in platyhelminths and mammals, which are also Grx–TrxR domain fusion proteins, the TrxRs from the filarial nematodes lacked glutathione disulfide reductase and Grx activities. The structural determinations revealed that the Grx domain of TrxR from B. malayi contains a cysteine (C22), conserved in TrxRs from clade IIIc nematodes, that directly interacts with the C-terminal cysteine-selenocysteine motif of the homo-dimeric subunit. Interestingly, despite this finding we found that altering C22 by mutation to serine did not affect enzyme catalysis. Thus, although the function of the Grx domain in these filarial TrxRs remains to be determined, the results obtained provide insights on key properties of this important family of selenoprotein flavoenzymes that are potential drug targets for treatment of filariasis.

Efficient flavinylation of glycosomal fumarate reductase by its own ApbE domain in Trypanosoma brucei.

A subset of flavoproteins has a covalently attached flavin prosthetic group enzymatically attached via phosphoester bonding. In prokaryotes, this is catalysed by alternative pyrimidine biosynthesis E (ApbE) flavin transferases. ApbE-like domains are present in few eukaryotic taxa, for example the N-terminal domain of fumarate reductase (FRD) of Trypanosoma, a parasitic protist known as a tropical pathogen causing African sleeping sickness. We use the versatile reverse genetic tools available for Trypanosoma to investigate the flavinylation of glycosomal FRD (FRDg) invivo in the physiological and organellar context. Using direct in-gel fluorescence detection of covalently attached flavin as proxy for activity, we show that the ApbE-like domain of FRDg has flavin transferase activity invivo. The ApbE domain is preceded by a consensus flavinylation target motif at the extreme N terminus of FRDg, and serine 9 in this motif is essential as flavin acceptor. The preferred mode of flavinylation in the glycosome was addressed by stoichiometric expression and comparison of native and catalytically inactive ApbE domains. In addition to the trans-flavinylation activity, the ApbE domain catalyses the intramolecular cis-flavinylation with at least fivefold higher efficiency. We discuss how the higher efficiency due to unusual fusion of the ApbE domain to its substrate protein FRD may provide a selective advantage by faster FRD biogenesis during rapid metabolic adaptation of trypanosomes. The first 37 amino acids of FRDg, including the consensus motif, are sufficient as flavinylation target upon fusion to other proteins. We propose FRDg(1-37) as 4-kDa heat-stable, detergent-resistant fluorescent protein tag and suggest its use as a new tool to study glycosomal protein import.

Non-secretory multiple myeloma with unusual TFG-ALK fusion showed dramatic response to ALK inhibition

Non-secretory multiple myeloma (NSMM) constitutes a distinct entity of multiple myeloma characterized by the absence of detectable monoclonal protein and rarely an absence of free light chains in the serum and urine. Given its rarity, the genomic landscape, clinical course, and prognosis of NSSM are not well characterized. Here, we report a case of a patient with relapsed and refractory NSMM with brain metastasis harboring a TFG-ALK fusion showing a dramatic and durable (over two years) response to commercially available anaplastic lymphoma kinase (ALK) inhibitors. The case emphasizes the beneficial role of molecular profiling in this target-poor disease.

Pericallosal berry aneurysm associated with azygous anterior cerebral artery – a case report

Introduction. Azygos anterior cerebral artery is a rare variant of the anterior segment of the circle of Willis caused by an unusual fusion of the normally paired A2 segments of the anterior cerebral artery (ACA). Despite its rare occurrence, it is associated with various vascular and structural cerebral abnormalities, particularly berry aneurysms. Aim. We aimed to present a case of a 41-year-old female patient who presented to our neurology department with complaints of headache. Description of the case. She had a positive paternal history of aneurysmal subarachnoid hemorrhage. Magnetic resonance angiography (MRA) of her brain revealed an azygos ACA (bifurcating into two pericallosal arteries) which was associated with a saccular aneurysm at its bifurcation point. She was referred to the interventional radiology department for preventive endovascular treatment. Conclusion. Azygos ACA carries a high risk of aneurysm development and its occlusion can potentially compromise blood supply to both cerebral hemispheres. It is therefore crucial for clinicians to be aware of its significance and to report its presence in angiographic studies.

United Kingdom lights up its unusual fusion reactor

United Kingdom lights up its unusual fusion reactor

Fuse to defuse: a self-limiting ribonuclease-ring nuclease fusion for type III CRISPR defence

Type III CRISPR systems synthesise cyclic oligoadenylate (cOA) second messengers in response to viral infection of bacteria and archaea, potentiating an immune response by binding and activating ancillary effector nucleases such as Csx1. As these effectors are not specific for invading nucleic acids, a prolonged activation can result in cell dormancy or death. Some archaeal species encode a specialised ring nuclease enzyme (Crn1) to degrade cyclic tetra-adenylate (cA4) and deactivate the ancillary nucleases. Some archaeal viruses and bacteriophage encode a potent ring nuclease anti-CRISPR, AcrIII-1, to rapidly degrade cA4 and neutralise immunity. Homologues of this enzyme (named Crn2) exist in type III CRISPR systems but are uncharacterised. Here we describe an unusual fusion between cA4-activated CRISPR ribonuclease (Csx1) and a cA4-degrading ring nuclease (Crn2) from Marinitoga piezophila. The protein has two binding sites that compete for the cA4 ligand, a canonical cA4-activated ribonuclease activity in the Csx1 domain and a potent cA4 ring nuclease activity in the C-terminal Crn2 domain. The cA4 binding affinities and activities of the two constituent enzymes in the fusion protein may have evolved to ensure a robust but time-limited cOA-activated ribonuclease activity that is finely tuned to cA4 levels as a second messenger of infection.

Abstract LB-B08: Identification and characterization of an unusual BRAF fusion oncoprotein with retained autoinhibitory domains

Abstract Fusion proteins involving the BRAF serine/threonine kinase occur in many cancers. Oncogenic BRAF fusion proteins usually consist of the BRAF kinase domain and an N-terminal fusion partner that replaces the critical domains required for BRAF autoinhibition. We applied whole-exome and RNA sequencing in a patient with glioblastoma multiforme (GBM) to identify a rearrangement between TTYH3, encoding a membrane-resident, calcium-activated chloride channel, and BRAF intron 1, resulting in a TTYH3-BRAF fusion protein that retained all structural prerequisites for BRAF autoinhibition. Indeed, the BRAF moiety of the fusion protein alone, which represented near-full-length BRAF without the amino acids encoded by exon 1 (BRAFΔE1), did not induce MEK/ERK phosphorylation or cellular transformation. Similarly, neither the TTYH3 moiety of the fusion protein nor full-length TTYH3 provoked ERK pathway activity or transformation. In contrast, TTYH3-BRAF displayed increased MEK phosphorylation potential and transforming activity, which were caused by TTYH3-mediated tethering of BRAFΔE1 to the (endo)membrane system. Consistent with this mechanism, a synthetic approach in which BRAFΔE1 was localized to the membrane by fusing it to the cytoplasmic tail of CD8 also induced transformation. Furthermore, we demonstrate that TTYH3-BRAF signals largely independent of a functional RAS-binding domain, but relies on an intact BRAF dimer interface and forms homo- and heterodimers with RAF1 or ARAF. Moreover, the MEK phosphorylation and transformation potential of TTYH3-BRAF requires the activation loop phosphorylation sites T599 and S602. Various cell line models, including primary human astrocytes and a GBM stem cell line, expressing TTYH3-BRAF exhibited increased MEK/ERK signaling. TTYH3-BRAF-induced MEK/ERK phosphorylation was blocked by clinically achievable concentrations of sorafenib, trametinib, and the paradox breaker PLX8394. These data provide the first example of a fully autoinhibited BRAF protein whose oncogenic potential is dictated by a distinct fusion partner and not by a structural change in BRAF itself. Citation Format: Florian Weinberg, Ricarda Herr, Martina Fröhlich, Christoph Heining, Sandra Braun, Corinna Spohr, Mary Iconomou, Viola Hollek, Michael Röring, Peter Horak, Simon Kreutzfeldt, Gregor Warsow, Barbara Hutter, Sebastian Uhrig, Olaf Neumann, David Reuss, Dieter Hendrik Heiland, Christof von Kalle, Wilko Weichert, Albrecht Stenzinger, Benedikt Brors, Hanno Glimm, Stefan Fröhling, Tilman Brummer. Identification and characterization of an unusual BRAF fusion oncoprotein with retained autoinhibitory domains [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr LB-B08. doi:10.1158/1535-7163.TARG-19-LB-B08

Unusual Fusion of α-Fluorinated Benzophenones under McMurry Reaction Conditions.

By exposure of α-fluorinated benzophenones to McMurry reaction conditions, we have observed the remarkable formation of 9,10-diphenylanthracene derivatives. This unexpected transformation necessitates the cleavage of the exceptionally stable aromatic C-F bond under mild McMurry conditions. In this work, the condensation of several related fluorinated benzo- and acetophenones has been investigated, which allow us to propose a domino-like fusion mechanism for this unusual transformation. The scope and limitations of the fluorine-promoted benzophenone fusion are subsequently discussed.

Structural Basis for Rare Earth Element Recognition by Methylobacterium extorquens Lanmodulin.

Lanmodulin (LanM) is a high-affinity lanthanide (Ln)-binding protein recently identified in Methylobacterium extorquens, a bacterium that requires Lns for the function of at least two enzymes. LanM possesses four EF-hands, metal coordination motifs generally associated with CaII binding, but it undergoes a metal-dependent conformational change with a 100 million-fold selectivity for LnIIIs and YIII over CaII. Here we present the nuclear magnetic resonance solution structure of LanM complexed with YIII. This structure reveals that LanM features an unusual fusion of adjacent EF-hands, resulting in a compact fold to the best of our knowledge unique among EF-hand-containing proteins. It also supports the importance of an additional carboxylate ligand in contributing to the protein's picomolar affinity for LnIIIs, and it suggests a role of unusual N i+1-H···N i hydrogen bonds, in which LanM's unique EF-hand proline residues are engaged, in selective LnIII recognition. This work sets the stage for a detailed mechanistic understanding of LanM's Ln selectivity, which may inspire new strategies for binding, detecting, and sequestering these technologically important metals.

Tridecanuclear CuII11Na2 Cagelike Silsesquioxanes

A series of three unprecedented heterometallic copper(II) sodium silsesquioxanes were isolated (i) via the unusual rearrangement process during synthesis of coordination polymers or (ii) via the self-assembly reaction using 2,2′-bipyridine. The unique type of these products’ molecular topology consists of an unusual fusion of two sandwich-like components (each including five copper and one sodium sites) via a central copper ion. These compounds correspond to the highest nuclearity among Cu(II)-based cage silsesquioxanes reported to date.

Sesquiterpene Synthase-3-Hydroxy-3-Methylglutaryl Coenzyme A Synthase Fusion Protein Responsible for Hirsutene Biosynthesis in Stereum hirsutum.

The wood-rotting mushroom Stereum hirsutum is a known producer of a large number of namesake hirsutenoids, many with important bioactivities. Hirsutenoids form a structurally diverse and distinct class of sesquiterpenoids. No genes involved in hirsutenoid biosynthesis have yet been identified or their enzymes characterized. Here, we describe the cloning and functional characterization of a hirsutene synthase as an unexpected fusion protein of a sesquiterpene synthase (STS) with a C-terminal 3-hydroxy-3-methylglutaryl-coenzyme A (3-hydroxy-3-methylglutaryl-CoA) synthase (HMGS) domain. Both the full-length fusion protein and truncated STS domain are highly product-specific 1,11-cyclizing STS enzymes with kinetic properties typical of STSs. Complementation studies in Saccharomyces cerevisiae confirmed that the HMGS domain is also functional in vivo Phylogenetic analysis shows that the hirsutene synthase domain does not form a clade with other previously characterized sesquiterpene synthases from Basidiomycota. Comparative gene structure analysis of this hirsutene synthase with characterized fungal enzymes reveals a significantly higher intron density, suggesting that this enzyme may be acquired by horizontal gene transfer. In contrast, the HMGS domain is clearly related to other fungal homologs. This STS-HMGS fusion protein is part of a biosynthetic gene cluster that includes P450s and oxidases that are expressed and could be cloned from cDNA. Finally, this unusual fusion of a terpene synthase to an HMGS domain, which is not generally recognized as a key regulatory enzyme of the mevalonate isoprenoid precursor pathway, led to the identification of additional HMGS duplications in many fungal genomes, including the localization of HMGSs in other predicted sesquiterpenoid biosynthetic gene clusters.IMPORTANCE Hirsutenoids represent a structurally diverse class of bioactive sesquiterpenoids isolated from fungi. Identification of their biosynthetic pathways will provide access to this chemodiversity for the discovery and synthesis of molecules with new bioactivities. The identification and successful cloning of the previously elusive hirsutene synthase from the S. hirsutum provide important insights and strategies for biosynthetic gene discovery in Basidiomycota. The finding of a terpene synthase-HMGS fusion, the discovery of other sesquiterpenoid biosynthetic gene clusters with dedicated HMGS genes, and HMGS gene duplications in fungal genomes give new importance to the role of HMGS as a key regulatory enzyme in isoprenoid and sterol biosynthesis that should be exploited for metabolic engineering.

Discovery of C-Glycosylpyranonaphthoquinones in Streptomyces sp. MBT76 by a Combined NMR-Based Metabolomics and Bioinformatics Workflow.

Mining of microbial genomes has revealed that actinomycetes harbor far more biosynthetic potential for bioactive natural products than anticipated. Activation of (cryptic) biosynthetic gene clusters and identification of the corresponding metabolites has become a focal point for drug discovery. Here, we applied NMR-based metabolomics combined with bioinformatics to identify novel C-glycosylpyranonaphthoquinones in Streptomyces sp. MBT76 and to elucidate the biosynthetic pathway. Following activation of the cryptic qin gene cluster for a type II polyketide synthase (PKS) by constitutive expression of its pathway-specific activator, bioinformatics coupled to NMR profiling facilitated the chromatographic isolation and structural elucidation of qinimycins A–C (1–3). The intriguing structural features of the qinimycins, including 8-C-glycosylation, 5,14-epoxidation, and 13-hydroxylation, distinguished these molecules from the model pyranonaphthoquinones actinorhodin, medermycin, and granaticin. Another novelty lies in the unusual fusion of a deoxyaminosugar to the pyranonaphthoquinone backbone during biosynthesis of the antibiotics BE-54238 A and B (4, 5). Qinimycins showed weak antimicrobial activity against Gram-positive bacteria. Our work shows the utility of combining bioinformatics, targeted activation of cryptic gene clusters, and NMR-based metabolic profiling as an effective pipeline for the discovery of microbial natural products with distinctive skeletons.

Unusual Fusion Proteins of HIV-1.

Despite its small genome size, the Human Immunodeficiency Virus 1 (HIV-1) is one of the most successful pathogens and has infected more than 70 million people worldwide within the last decades. In total, HIV-1 expresses 16 canonical proteins from only nine genes within its 10 kb genome. Expression of the structural genes gag, pol, and env, the regulatory genes rev and tat and the accessory genes vpu, nef, vpr, and vif enables assembly of the viral particle, regulates viral gene transcription, and equips the virus to evade or counteract host immune responses. In addition to the canonically expressed proteins, a growing number of publications describe the existence of non-canonical fusion proteins in HIV-1 infected cells. Most of them are encoded by the tat-env-rev locus. While the majority of these fusion proteins (e.g., TNV/p28tev, p186Drev, Tat1-Rev2, Tat^8c, p17tev, or Ref) are the result of alternative splicing events, Tat-T/Vpt is produced upon programmed ribosomal frameshifting, and a Rev1-Vpu fusion protein is expressed due to a nucleotide polymorphism that is unique to certain HIV-1 clade A and C strains. A better understanding of the expression and activity of these non-canonical viral proteins will help to dissect their potential role in viral replication and reveal how HIV-1 optimized the coding potential of its genes. The goal of this review is to provide an overview of previously described HIV-1 fusion proteins and to summarize our current knowledge of their expression patterns and putative functions.

Lectotypifications in Orthoclada (Poaceae, Panicoideae, Zeugiteae)

The grass genus Orthoclada Beauvois (1812: 69) is currently placed in the subfamily Panicoideae, tribe Zeugiteae (Soreng et al. 2015), and includes only two species from forests of Tropical America and Africa: O. laxa (Richard 1792: 106) Beauvois (1812: 70) and O. africana Hubbard (1940: 3419) (Kellogg 2015, The Plant List 2016). The genus was formerly included in subfamily Centothecoideae (Clayton & Renvoize 1986), which was recently submerged into the Panicoideae (Sánchez-Ken & Clark 2010, Soreng et al. 2015). Orthoclada is characterized by its bisexual flowers and the unusual fusion of its rachilla internode to the palea keels (Kellogg 2015).