Coiled-coil Dimerization Domain Research Articles

Swine acute diarrhea syndrome coronavirus nucleocapsid protein antagonizes the IFN response through inhibiting TRIM25 oligomerization and functional activation of RIG-I/TRIM25.

Swine acute diarrhea syndrome coronavirus (SADS-CoV), an emerging Alpha-coronavirus, brings huge economic loss in swine industry. Interferons (IFNs) participate in a frontline antiviral defense mechanism triggering the activation of numerous downstream antiviral genes. Here, we demonstrated that TRIM25 overexpression significantly inhibited SADS-CoV replication, whereas TRIM25 deficiency markedly increased viral yield. We found that SADS-CoV N protein suppressed interferon-beta (IFN-β) production induced by Sendai virus (SeV) or poly(I:C). Moreover, we determined that SADS-CoV N protein interacted with RIG-I N-terminal two caspase activation and recruitment domains (2CARDs) and TRIM25 coiled-coil dimerization (CCD) domain. The interaction of SADS-CoV N protein with RIG-I and TRIM25 caused TRIM25 multimerization inhibition, the RIG-I-TRIM25 interaction disruption, and consequent the IRF3 and TBK1 phosphorylation impediment. Overexpression of SADS-CoV N protein facilitated the replication of VSV-GFP by suppressing IFN-β production. Our results demonstrate that SADS-CoV N suppresses the host IFN response, thus highlighting the significant involvement of TRIM25 in regulating antiviral immune defenses.

Structural and biophysical characterization of PadR family protein Rv1176c of Mycobacterium tuberculosis H37Rv

Rv1176c of Mycobacterium tuberculosis H37Rv belongs to the PadR-s1 subfamily of the PadR family of protein. Rv1176c forms a stable dimer in solution. Its stability is characterized by a thermal melting transition temperature (Tm) of 39.4 °C. The crystal structure of Rv1176c was determined at a resolution of 2.94 Å, with two monomers in the asymmetric unit. Each monomer has a characteristic N-terminal winged-helix-turn-helix DNA-binding domain. Rv1176c C-terminal is a coiled-coil dimerization domain formed of α-helices α5 to α7. In the Rv1176c dimer, there is domain-swapping of the C-terminal domain in comparison to other PadR homologs. In the dimer, there is a long inter-subunit tunnel in which different ligands can bind. Rv1176c was found to bind to the promoter region of its own gene with high specificity. M. smegmatis MC2 155 genome lacks homolog of Rv1176c. Therefore, it was used as a surrogate to characterize the functional role of Rv1176c. Expression of Rv1176c in M. smegmatis MC2 155 cells imparted enhanced tolerance towards oxidative stress. Rv1176c expressing M. smegmatis MC2 155 cells exhibited enhanced intracellular survival in J774A.1 murine macrophage cells. Overall, our studies demonstrate Rv1176c to be a PadR-s1 subfamily transcription factor that can moderate the effect of oxidative stress.

Role of GARP family transcription factors in the regulatory network for nitrogen and phosphorus acquisition

The GARP (Golden2, ARR-B, Psr1) family proteins with a conserved DNA-binding domain, called the B-motif, are plant-specific transcription factors involved in the regulation of various physiological processes. The GARP family proteins are divided into members that function as monomeric transcription factors, and members that function as transcription factors in the dimeric form, owing to the presence of a coiled-coil dimerization domain. Recent studies revealed that the dimer-forming GARP family members, which are further divided into the PHR1 and NIGT1 subfamilies, play critical roles in the regulation of phosphorus (P) and nitrogen (N) acquisition. In this review, we present a general overview of the GARP family proteins and discuss how several members of the PHR1 and NIGT1 subfamilies are involved in the coordinated acquisition of P and N in response to changes in environmental nutrient conditions, while mainly focusing on the recent findings that enhance our knowledge of the roles of PHR1 and NIGT1 in phosphate starvation signaling and nitrate signaling.

Intrinsic mesoscale properties of a Polycomb protein underpin heterochromatin fidelity.

Little is understood about how the two major types of heterochromatin domains (HP1 and Polycomb) are kept separate. In the yeast Cryptococcus neoformans, the Polycomb-like protein Ccc1 prevents deposition of H3K27me3 at HP1 domains. Here we show that phase separation propensity underpins Ccc1 function. Mutations of the two basic clusters in the intrinsically disordered region or deletion of the coiled-coil dimerization domain alter phase separation behavior of Ccc1 in vitro and have commensurate effects on formation of Ccc1 condensates in vivo, which are enriched for PRC2. Notably, mutations that alter phase separation trigger ectopic H3K27me3 at HP1 domains. Supporting a direct condensate-driven mechanism for fidelity, Ccc1 droplets efficiently concentrate recombinant C. neoformans PRC2 in vitro whereas HP1 droplets do so only weakly. These studies establish a biochemical basis for chromatin regulation in which mesoscale biophysical properties play a key functional role.

Cytoskeletal regulation of a transcription factor by DNA mimicry via coiled-coil interactions.

A long-established strategy for transcription regulation is the tethering of transcription factors to cellular membranes. By contrast, the principal effectors of Hedgehog signalling, the GLI transcription factors, are regulated by microtubules in the primary cilium and the cytoplasm. How GLI is tethered to microtubules remains unclear. Here, we uncover DNA mimicry by the ciliary kinesin KIF7 as a mechanism for the recruitment of GLI to microtubules, wherein the coiled-coil dimerization domain of KIF7, characterized by its striking shape, size and charge similarity to DNA, forms a complex with the DNA-binding zinc fingers in GLI, thus revealing a mode of tethering a DNA-binding protein to the cytoskeleton. GLI increases KIF7 microtubule affinity and consequently modulates the localization of both proteins to microtubules and the cilium tip. Thus, the kinesin-microtubule system is not a passive GLI tether but a regulatable platform tuned by the kinesin-transcription factor interaction. We retooled this coiled-coil-based GLI-KIF7 interaction to inhibit the nuclear and cilium localization of GLI. This strategy can potentially be exploited to downregulate erroneously activated GLI in human cancers.

Abstract 5933: Biochemical characterization of FGFR2-PPHLN1 and BCR-FGFR1, drivers of epithelial and hematological malignancies

Abstract Fibroblast growth factor receptors (FGFRs) are part of the receptor tyrosine kinase (RTK) family and are essential in the activation of various downstream signaling pathways, which are necessary for cell differentiation and proliferation. However, mutation and translocation of FGFRs leads to aberrant activation of signaling, which often results in cancer. This work focuses on the FGFR2-PPHLN1 fusion protein, a driver of intrahepatic cholangiocarcinoma (ICC), and the BCR-FGFR1 fusion protein, a driver of stem cell leukemia/lymphoma (SCLL). Both FGFR2-PPHLN1 and BCR-FGFR1 are poorly characterized, resulting in few therapies and clinical advancements for patients positive for these oncogene-driven neoplasms. Here, we aim to characterize these two FGFR fusion proteins, and analyze therapeutic options to treat these malignancies. Both FGFR2-PPHLN1 and BCR-FGFR1 contain a coiled-coil dimerization domain fused to a constitutively activated kinase provided by either FGFR2 or FGFR1, respectively. This work shows the reliance of these FGFR fusion proteins on the tyrosine kinase activity of the respective FGFR, as kinase dead mutants were not biologically active. Additionally both FGFR2-PPHLN1 and BCR-FGFR1 expressing cells exhibited an over activation of the RAS/MAPK and JAK/STAT pathways, indicating the importance of these pathways in FGFR1-PPHLN1 driven ICC, and BCR-FGFR1 driven SCLL, respectively. Transformed cells expressing FGFR2-PPHLN1 are sensitive to FGFR inhibitor BGJ398. However, this sensitivity is abolished when a kinase activating mutation is introduced with FGFR2(N549K)-PPHLN1. Residue N549 in the FGFR2 kinase domain functions as part of a molecular brake; mutation of this residue disrupts the auto-inhibitory network of hydrogen bonds, leading to increased kinase activation. The loss in sensitivity to BJG398 treatment for cells expressing FGFR2(N549K)-PPHLN1 indicates that BGJ398 treatment alone may be ineffective once secondary mutations are established in the kinase domain. Preliminary data suggests that transformed cells expressing either FGFR2-PPHLN1, or kinase activated FGFR2(N549K)-PPHLN1, may be sensitive to treatment with FGFR inhibitor TAS-120, suggesting that TAS-120 treatment could be beneficial for patients with FGFR2-PPHLN1 driven ICC. Furthermore, we establish BCR-FGFR1 as a client of the Hsp90 chaperone complex. Transformed cells expressing BCR-FGFR1 are sensitive to the Hsp90 inhibitor, Ganetespib, and also respond to combined treatment with Ganetespib and FGFR inhibitor BGJ398, suggesting novel clinical treatment options for patients positive for this fusion. Collectively, we show that the tyrosine kinase activity provided by FGFR2 or FGFR1, respectively is required for the biological activity of these fusion proteins, and we establish novel treatment options for patients with FGFR2-PPHLN1 or BCR-FGFR1 driven malignancies. Citation Format: Malalage Nicole Peiris, Fangda Li, April N. Meyer, Dalida Warda, Daniel J. Donoghue. Biochemical characterization of FGFR2-PPHLN1 and BCR-FGFR1, drivers of epithelial and hematological malignancies [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5933.

Ets1 Plays a Critical Role in MLL/EB1-Mediated Leukemic Transformation in a Mouse Bone Marrow Transplantation Model

Leukemogenic potential of MLL fusion with the coiled-coil domain-containing partner genes and the downstream target genes of this type of MLL fusion have not been clearly investigated. In this study, we demonstrated that the coiled-coil–four-helix bundle structure of EB1 that participated in the MLL/EB1 was required for immortalizing mouse bone marrow (BM) cells and producing myeloid, but not lymphoid, cell lines. Compared to MLL/AF10, MLL/EB1 had low leukemogenic ability. The MLL/EB1 cells grew more slowly owing to increased apoptosis in vitro and induced acute monocytic leukemia with an incomplete penetrance and longer survival in vivo. A comparative analysis of transcriptome profiling between MLL/EB1 and MLL/AF10 cell lines revealed that there was an at least two-fold difference in the induction of 318 genes; overall, 51.3% (163/318) of the genes were known to be bound by MLL, while 15.4% (49/318) were bound by both MLL and MLL/AF9. Analysis of the 318 genes using Gene Ontology–PANTHER overrepresentation test revealed significant differences in several biological processes, including cell differentiation, proliferation/programmed cell death, and cell homing/recruitment. The Ets1 gene, bound by MLL and MLL/AF9, was involved in several biological processes. We demonstrated that Ets1 was selectively upregulated by MLL/EB1. Short hairpin RNA knockdown of Ets1 in MLL/EB1 cells reduced the expression of CD115, apoptosis rate, competitive engraftment to BM and spleen, and incidence of leukemia and prolonged the survival of the diseased mice. Our results demonstrated that MLL/EB1 upregulated Ets1, which controlled the balance of leukemia cells between apoptosis and BM engraftment/clonal expansion.Novelty and impact of this studyThe leukemogenic potential of MLL fusion with cytoplasmic proteins containing coiled-coil dimerization domains and the downstream target genes of this type of MLL fusion remain largely unknown. Using a retroviral transduction/transplantation mouse model, we demonstrated that MLL fusion with the coiled-coil–four-helix bundle structure of EB1 has low leukemogenic ability; Ets1, which is upregulated by MLL/EB1, plays a critical role in leukemic transformation by balance between apoptosis and BM engraftment/clonal expansion.

The MerR-like protein BldC binds DNA direct repeats as cooperative multimers to regulate Streptomyces development

Streptomycetes are notable for their complex life cycle and production of most clinically important antibiotics. A key factor that controls entry into development and the onset of antibiotic production is the 68-residue protein, BldC. BldC is a putative DNA-binding protein related to MerR regulators, but lacks coiled-coil dimerization and effector-binding domains characteristic of classical MerR proteins. Hence, the molecular function of the protein has been unclear. Here we show that BldC is indeed a DNA-binding protein and controls a regulon that includes other key developmental regulators. Intriguingly, BldC DNA-binding sites vary significantly in length. Our BldC-DNA structures explain this DNA-binding capability by revealing that BldC utilizes a DNA-binding mode distinct from MerR and other known regulators, involving asymmetric head-to-tail oligomerization on DNA direct repeats that results in dramatic DNA distortion. Notably, BldC-like proteins radiate throughout eubacteria, establishing BldC as the founding member of a new structural family of regulators.

Cep120 promotes microtubule formation through a unique tubulin binding C2 domain.

Centrioles are microtubule-based structures that play essential roles in cell division and cilia biogenesis. Cep120 is an important protein for correct centriole formation and mutations in the Cep120 gene cause severe human diseases like Joubert syndrome and complex ciliopathies. Here, we show that Cep120 contains three consecutive C2 domains that are followed by a coiled-coil dimerization domain. Surprisingly, unlike the classical C2 domains, all three Cep120 C2 domains lack calcium- and phospholipid-binding activities. However, biophysical and biochemical assays revealed that the N-terminal Cep120 C2 domain (C2A) binds to both tubulin and microtubules, and promotes microtubule formation. Structural analyses coupled with mutagenesis identified a highly conserved, positively charged residue patch on the surface of Cep120 C2A, which mediates the interaction with tubulin and microtubules. Together, our results establish Cep120 C2A as a unique microtubule-binding domain. They further provide insights into the molecular mechanism of Cep120 during centriole biogenesis.

Structure of the WD40-domain of human ATG16L1.

Autophagy-related protein ATG16L1 is a component of the mammalian ATG12∼ATG5/ATG16L1 complex, which acts as E3-ligase to catalyze lipidation of LC3 during autophagosome biogenesis. The N-terminal part of ATG16L1 comprises the ATG5-binding site and coiled-coil dimerization domain, both also present in yeast ATG16 and essential for bulk and starvation induced autophagy. While absent in yeast ATG16, mammalian ATG16L1 further contains a predicted C-terminal WD40-domain, which has been shown to be involved in mediating interaction with diverse factors in the context of alternative functions of autophagy, such as inflammatory control and xenophagy. In this work, we provide detailed information on the domain boundaries of the WD40-domain of human ATG16L1 and present its crystal structure at a resolution of 1.55 Å.

Phosphorylation of HEXIM1 at Tyr271 and Tyr274 Promotes Release of P-TEFb from the 7SK snRNP Complex and Enhances Proviral HIV Gene Expression.

Efficient HIV transcription requires P-TEFb, an essential co-factor for Tat. In actively replicating cells, P-TEFb is incorporated into the 7SK snRNP complex together with the repressor protein HEXIM1. Using an affinity purification-tandem mass spectrometry approach to identify modification sites on HEXIM1 that regulate the sequestration of P-TEFb by 7SK snRNP, we found that HEXIM1 can be phosphorylated on adjacent residues in a region immediately upstream of the coiled-coil dimerization domain (Ser268, Thr270, Tyr271, and Tyr274). Phosphomimetic mutations of Tyr271 and Tyr274 disrupted the assembly of P-TEFb and HEXIM1 into the 7SK snRNP complex. Although Y271E/Y274E did not adversely affect the nuclear localization pattern of HEXIM1, it induced the redistribution of the CDK9 subunit of P-TEFb into the cytoplasm. By contrast, the Y271F/Y274F HEXIM1 mutant assembled normally with P-TEFb within the 7SK snRNP complex but severely reduced proviral gene expression in T cells in response to activation signals and caused a severe growth defect of Jurkat T cells. Thus, Y271F/Y274F, which cannot be phosphorylated on these residues, appears to block the exchange of active P-TEFb from the 7SK complex, thereby limiting the level of P-TEFb below the threshold required to support transcription elongation of the HIV provirus and cellular genes.

CGMP-dependent protein kinase Iβ phosphorylates and regulates the function of the actin/myosin-associatedprotein caldesmon

Background The type I cGMP-dependent protein kinases (PKGIa and PKGIb) are splice variants that differ in their first ~100 amino acids, giving each isoform unique dimerization and autoinhibitory domains. The unique coiled-coil dimerization domains mediate isoform specific proteinprotein interactions, and we have previously identified the amino acids that are important in mediating the interaction between PKGIb and its two known interaction partners, TFII-I and IRAG [1].

Lever Arm Mobility and Force Generation in Ncd, a Minus-End Kinesin Motor

Unlike the conventional kinesin motor proteins, which step towards the plus end of the microtubule, the kinesin-14 family translocates cargo towards microtubule minus ends. Following microtubule attachment, kinesin-14 motors use energy derived from ATP hydrolysis to swing a arm, consisting of a long coiled-coil dimerization domain, in order to move their cargo in the direction of travel. While atomic-resolution structures of several kinesin-14 constructs have been solved, the active states of force transduction only occur when the motor is attached to the microtubule. Thus, the mechanism that drives lever arm movement remains unclear. We solved a series of sub-nanometer cryo-EM reconstructions of dimeric ncd, a kinesin-14 family member, in different chemical states while attached to the microtubule. The maps reveal a series of well-defined structural rearrangements in the ncd motor domain, suggesting a mechanism for how microtubule attachment and subsequent ATP binding can trigger a lever arm swing. Unexpectedly, our maps also indicate that the unbound (or “tethered”) catalytic head of our dimeric ncd construct, which does not contact the microtubule, remains rotationally mobile in all the microtubule-attached nucleotide states we examined. In contrast, the other head always attaches to the microtubule in a well-defined orientation, and the lever arm of the motor assembly projects rigidly away from this head while exhibiting either “pre-” or “post-” power stroke conformations. These results suggest a model in which the catalytic domains of an unattached ncd dimer oscillate between different lever arm states in order to facilitate the search for a new microtubule binding site; attachment would then lock the lever arm angle in preparation for a power stroke.

Myeloproliferative Neoplasm with BCR-JAK2 Fusion Gene As the Result of t(9;22)(p24,11.2) in a Brazilian Patient

AbstractAbstract 4808 IntroductionChronic Myeloid Leukemia (CML) is the most well described disease result of t(9;22)(q34,q11.2). This chromosomal rearrangement leads to well-know BCR-ABL fusion that promotes tyrosine kinase activity. There are others oncogenic BCR fusion found such as PDGFRA (4q12), FGFR1(8p12) that causes myeloproliferative disorders (MD).JAK2 gene is one of the 4 genes members of JAK family. The JAK2 V617F mutation which results from a G –>T transversion at nucleotide 1849 in exon 14 of the JAK2 gene, the consequence of which is substitution of valine by phenylalanine at codon 617 is associated with MD and it is a major diagnosis criterion for Primary Myeloficrosis, Polycythaemia vera and Essential thrombocytemia. There are described a lot chromosomal translocations involving the JAK2 locus.We report an extremely rare case with BCR-JAK2 fusion gene as the result of t(9;22)(p24,q11.2) for the first time in Brazilian people, and it is the 6thcase all of the world. Case ReportIn April 2010, a 54 years old male patient presented fatigue, abdominal pain and splenomegaly. A blood count revealed leukocytosis 93.380/mm3 with a predominance of neutrophils and left shift. Conventional cytogenetic analysis was performed and it was evidenced 46,XY, t(9;22)(p24;q11.2) in 90% metaphases examined, due to expected association it was promoted BCR-ABL1 fusion gene and it was not detected by using RT-PCR. He was treated with imatinib 400mg/day because the involvement of BCR gene. After three months he presented weight loss, progressive splenomegaly without hematologic response and it was modified to Dasatinib 150mg/day plus hydroxyureia 3g/day. In August 2011, due to not hematologic response, it was stopped Dasatinib treatment and nowadays patient has been treating with hydroxyureia 1.5g/day. His last follow up in May 2012, blood count was abnormal Hb 16.8g/dl leukocytes 7730/mm3 and low platelets count 32.000/mm3. The differential count showed 65.3% segmented granulocytes, 13.6% eosinophlis, 1.6% basophil, 2.6% monocytes, 16.9% lymphocytes.It was repeated conventional Karyotyping and it was evidenced 46,XY, t(9;22)(p24;q11.2) in all of metaphases examined. The presence of BCR-ABL rearrangement was excluded by using the fluorescence in situ hybridization (FISH) using a BCR-ABL probe. In addition, it was not evidenced FIP1L1-PDGFRa fusion gene and JAK2 V617F mutation by using RT-PCR. DiscussionWe have described a male patient with MD with t(9;22)(p24;q11.2) wich leads to the BCR-JAK2 fusion and it was not evidenced BCR-ABL1, FIP1L1-PDGFRa fusion genes and JAK2 V617F mutation by using RT-PCR. Moreover, patient has not been achieved hematologic response with tyrosine kinase inhibitors: imatinib and dasatinib. In the five cases reported three presented MD, one Acute Myeloid Leukemia and one Acute Lymphoblastic Leukemia. Only in one case report it was prescribed imatinib and the patient lost the follow up (Table1). The BCR-JAK2 fusion protein contain the coiled-coil dimerization domain of BCR and the protein tyrosine Kinase domain (JH1) of JAK2. It was not possible to define what would be the best therapy, because tyrosine kinase inhibitors may not be effective to the BCR-JAK2 fusion. Maybe in MD presentation, we could return to pre- tyrosine kinase inhibitors era based on treatments with hydroxyureia, subcutaneous cytarabine and interferon for patients that were not potential candidates for allogeneic transplant.ReferencesAgesextranslocationClinical presentationTreatmentFollow upGriesing F et al63Ft(9;22)(p24;q11.2)MDHy; Cy; MitDeathCirmena G et al67Ft(9;22)(p24;q11)AMLHD + allo BMTDeathLane SW et al44Mt(9;22)(p24;q11.2)MDNot describedNot describedElnaggar MM et al84Mt(9;22)(p24;q11.2)MDHy; ImaLost follow upTirado CA et al14Mt(9;22)(p24;q11.2)ALLPolychemotherapyRemissionHy: Hydroxyurea; Cy: Cytarabine; Mit: Mitoxantrone; HD: High dose chemotherapy; allo BMT allogeneic Bone Marrow Transplantation; Ima: imatinib; ALL: Acute lymphoblastic leukemia. Disclosures:No relevant conflicts of interest to declare.

Toll/Interleukin-1 Receptor Domain Dimers as the Platform for Activation and Enhanced Inhibition of Toll-like Receptor Signaling

TIR (Toll/IL-1 receptor) domains mediate interactions between TLR (Toll-like) or IL-1 family receptors and signaling adapters. While homotypic TIR domain interactions mediate receptor activation they are also usurped by microbial TIR domain containing proteins for immunosuppression. Here we show the role of a dimerized TIR domain platform for the suppression as well as for the activation of MyD88 signaling pathway. Coiled-coil dimerization domain, present in many bacterial TCPs, potently augments suppression of TLR/IL-1R signaling. The addition of a strong coiled-coil dimerization domain conferred the superior inhibition against the wide spectrum of TLRs and prevented the constitutive activation by a dimeric TIR platform. We propose a molecular model of MyD88-mediated signaling based on the dimerization of TIR domains as the limiting step.

Functional self-assembling polypeptide bionanomaterials

Bionanotechnology seeks to modify and design new biopolymers and their applications and uses biological systems as cell factories for the production of nanomaterials. Molecular self-assembly as the main organizing principle of biological systems is also the driving force for the assembly of artificial bionanomaterials. Protein domains and peptides are particularly attractive as building blocks because of their ability to form complex three-dimensional assemblies from a combination of at least two oligomerization domains that have the oligomerization state of at least two and three respectively. In the present paper, we review the application of polypeptide-based material for the formation of material with nanometre-scale pores that can be used for the separation. Use of antiparallel coiled-coil dimerization domains introduces the possibility of modulation of pore size and chemical properties. Assembly or disassembly of bionanomaterials can be regulated by an external signal as demonstrated by the coumermycin-induced dimerization of the gyrase B domain which triggers the formation of polypeptide assembly.

The Pore of the Voltage-Gated Proton Channel

In classical tetrameric voltage-gated ion channels four voltage-sensing domains (VSDs), one from each subunit, control one ion permeation pathway formed by four pore domains. The human Hv1 proton channel has a different architecture, containinga VSD, but lacking a pore domain. Since its location is not known, we searched for the Hv permeation pathway. We find that mutation of the S4 segment's third arginine R211 (R3) compromises proton selectivity, enabling conduction of a metal cation and even of the large organic cation guanidinium, reminiscent of Shaker's omega pore. In the open state, R3 appears to interact with an aspartate (D112) that is situated in the middle of S1 and is unique to Hv channels. The double mutation of both residues further compromises cation selectivity. We propose that membrane depolarization reversibly positions R3 next to D112 inthe transmembrane VSD to form the ion selectivity filter in the channel's open conformation.

Two alternatively spliced 5′BCR/3′JAK2 fusion transcripts in a myeloproliferative neoplasm with a three-way t(9;18;22)(p23;p11.3;q11.2) translocation

Philadelphia (Ph)-negative myeloproliferative neoplasms (MPNs) are known to harbor alterations of the tyrosine kinase JAK2 (9p24), resulting in the constitutive autoactivation of the encoded protein. Here, we report an unclassifiable MPN case, BCR/ABL1-negative, showing a three-way t(9;18;22)(p23;p11.3;q11.2) translocation, which generates a 5'BCR/3'JAK2 gene by fusing BCR at intron 1 to JAK2 at intron 14 on the derivative chromosome 22. The fusion gene produced two alternatively spliced 5'BCR/3'JAK2 transcripts, fusing in-frame BCR exon 1 to JAK2 exon 15 and exon 17. This is the first report of the simultaneous occurrence of two BCR/JAK2 fusion transcripts in the same sample and of the longer transcript isoform (BCR exon 1 fused to JAK2 exon 15). Notably, both BCR/JAK2 encoded fusion proteins are predicted to juxtapose the coiled-coil dimerization domain of BCR to the catalytically inactive pseudokinase domain (JH2), entirely or partially deprived of the inhibitory region 1 (IR1). Interestingly, IR1 is involved in the auto-inhibitory interaction with the JAK2 kinase domain (JH1), which may result in deregulation of JAK2 activity.

Kinesins at a glance.

Kinesin was discovered in 1985 – 25 years ago – based on its motility in cytoplasm extruded from the giant axon of the squid ([Allen et al., 1982][1]; [Brady et al., 1982][2]; [Vale et al., 1985][3]). The purified protein, now known as kinesin-1, consisted of two heavy chains with motor activity

A green fluorescent protein solubility screen in E. coli reveals domain boundaries of the GTP‐binding domain in the P element transposase

Guanosine triphosphate (GTP) binding and hydrolysis events often act as molecular switches in proteins, modulating conformational changes between active and inactive states in many signaling molecules and transport systems. The P element transposase of Drosophila melanogaster requires GTP binding to proceed along its reaction pathway, following initial site-specific DNA binding. GTP binding is unique to P elements and may represent a novel form of transpositional regulation, allowing the bound transposase to find a second site, looping the transposon DNA for strand cleavage and excision. The GTP-binding activity has been previously mapped to the central portion of the transposase protein; however, the P element transposase contains little sequence identity with known GTP-binding folds. To identify soluble, active transposase domains, a GFP solubility screen was used testing the solubility of random P element gene fragments in E. coli. The screen produced a single clone spanning known GTP-binding residues in the central portion of the transposase coding region. This clone, amino acids 275-409 in the P element transposase, was soluble, highly expressed in E.coli and active for GTP-binding activity, therefore is a candidate for future biochemical and structural studies. In addition, the chimeric screen revealed a minimal N-terminal THAP DNA-binding domain attached to an extended leucine zipper coiled-coil dimerization domain in the P element transposase, precisely delineating the DNA-binding and dimerization activities on the primary sequence. This study highlights the use of a GFP-based solubility screen on a large multidomain protein to identify highly expressed, soluble truncated domain subregions.