Coiled-coil Dimers Research Articles

Axial Elasticity and Mechanical Fragmentation of Desmin Intermediate Filaments

Desmin is the intermediate filament of muscle cells. Desmin filaments are thought to play fundamental roles in cellular force transduction and the maintenance of structural integrity under mechanical exposure. Although the importance of desmin elasticity and assembly/disassembly dynamics in cellular mechanics is being increasingly recognized, the molecular basis of desmin's elasticity or its disassembly pathway are little understood. In the present work we explored the elasticity of purified desmin filaments by stretching them longitudinally with surface-tension forces.Desmin, purified from chicken gizzard, was polymerized by the addition of MgCl2. Polymerized filaments in a buffer droplet were then stretched by using a custom-built horizontal rotor assembly. Driven by centrifugal force, the droplet spread radially on freshly cleaved mica surface. Partially surface-adsorbed desmin filaments became extended by the receding meniscus and were captured on mica in the over-stretched state. The stretch force acting on the entire cross-sectional area of desmin was calculated to be 4 nN.As a result of this molecular combing, the average contour length of desmin filaments increased from 0.89 µm to 1.38 µm, which corresponds to a 1.6-fold axial stretch. Molecular combing together with EDTA-treatment caused the fragmentation of desmin filaments into short, 60- to 120-nm-long and 4-nm-wide structures displaying periodic, 34 nm-surface protrusions. Based on these calculations the observed fragments are hypothesized to be protofibril fragments composed of laterally attached desmin dimers. The orientation axis of the surface-constrained fragments deviated widely from the filament axis, suggesting that the protofibrils are not aligned in parallel within the filament during axial load. Furthermore, the emergence of protofibril fragments suggests that the head or tail domains of coiled-coil desmin dimers are the load-bearing elements during axial stretch.

Tropomyosin - master regulator of actin filament function in the cytoskeleton.

Tropomyosin (Tpm) isoforms are the master regulators of the functions of individual actin filaments in fungi and metazoans. Tpms are coiled-coil parallel dimers that form a head-to-tail polymer along the length of actin filaments. Yeast only has two Tpm isoforms, whereas mammals have over 40. Each cytoskeletal actin filament contains a homopolymer of Tpm homodimers, resulting in a filament of uniform Tpm composition along its length. Evidence for this 'master regulator' role is based on four core sets of observation. First, spatially and functionally distinct actin filaments contain different Tpm isoforms, and recent data suggest that members of the formin family of actin filament nucleators can specify which Tpm isoform is added to the growing actin filament. Second, Tpms regulate whole-organism physiology in terms of morphogenesis, cell proliferation, vesicle trafficking, biomechanics, glucose metabolism and organ size in an isoform-specific manner. Third, Tpms achieve these functional outputs by regulating the interaction of actin filaments with myosin motors and actin-binding proteins in an isoform-specific manner. Last, the assembly of complex structures, such as stress fibers and podosomes involves the collaboration of multiple types of actin filament specified by their Tpm composition. This allows the cell to specify actin filament function in time and space by simply specifying their Tpm isoform composition.

An Explicitly Solvated Full Atomistic Model of the Cardiac Thin Filament and Application on the Calcium Binding Affinity Effects from Familial Hypertrophic Cardiomyopathy Linked Mutations

The former version of our cardiac thin filament model consisted of the troponin complex (cTn), two coiled-coil dimers of tropomyosin (Tm), and twenty-nine actin subunits. We now present the newest revision of the model to include both solvation and ionization. The model was developed to continue our study of genetic mutations in the cardiac thin filament proteins which are linked to familial hypertrophic cardiomyopathies. Binding of calcium to the cardiac troponin C subunit (cTnC) causes subtle conformational changes to propagate through the cTnC to the inhibitor subunit (cTnI) which then detaches from actin. Conformational changes propagate through to the cTnT subunit, which allow for the movement of Tm into the open position along actin. Myosin heads can bind to the seven open binding sites on actin, which upon hydrolysis of ATP leads to muscle contraction. Calcium disassociation allows for the reverse to occur, which results in muscle relaxation. Alterations in the calcium binding affinity can disrupt the natural processes of the heart. The inclusion of explicit TIP3 water solvation and an ionic concentration of 0.15 mol/L allows for the model to mimic the true conditions that the cardiac thin filament would feel. The move from implicit to explicit solvation allows us to get better individual local solvent to protein interactions; which are important when observing the N-lobe calcium binding pocket of the cTnC. We are able to compare in silica and in vitro experimental results to better understand the physiological effects from mutants, such as the R92L/W and F110V/I of the cTnT, on the calcium binding affinity compared to the wild type.

Computational characterization of parallel dimeric and trimeric coiled-coils using effective amino acid indices.

The coiled-coil, which consists of two or more α-helices winding around each other, is a ubiquitous and the most frequently observed protein-protein interaction motif in nature. The coiled-coil is known for its straightforward heptad repeat pattern and can be readily recognized based on protein primary sequences, exhibiting a variety of oligomer states and topologies. Due to the stable interaction formed between their α-helices, coiled-coils have been under close scrutiny to design novel protein structures for potential applications in the fields of material science, synthetic biology and medicine. However, their broader application requires an in-depth and systematic analysis of the sequence-to-structure relationship of coiled-coil folding and oligomeric formation. In this article, we propose a new oligomerization state predictor, termed as RFCoil, which exploits the most useful and non-redundant amino acid indices combined with the machine learning algorithm - random forest (RF) - to predict the oligomeric states of coiled-coil regions. Benchmarking experiments show that RFCoil achieves an AUC (area under the ROC curve) of 0.849 on the 10-fold cross-validation test using the training dataset and 0.855 on the independent test using the validation dataset, respectively. Performance comparison results indicate that RFCoil outperforms the four existing predictors LOGICOIL, PrOCoil, SCORER 2.0 and Multicoil2. Furthermore, we extract a number of predominant rules from the trained RF model that underlie the oligomeric formation. We also present two case studies to illustrate the applicability of the extracted rules to the prediction of coiled-coil oligomerization state. The RFCoil web server, source codes and datasets are freely available for academic users at http://protein.cau.edu.cn/RFCoil/.

Exploring alternate states and oligomerization preferences of coiled-coils by de novo structure modeling.

Homomeric coiled-coils can self-assemble into a wide range of structural states with different helix topologies and oligomeric states. In this study, we have combined de novo structure modeling with stability calculations to simultaneously predict structure and oligomeric states of homomeric coiled-coils. For dimers an asymmetric modeling protocol was developed. Modeling without symmetry constraints showed that backbone asymmetry is important for the formation of parallel dimeric coiled-coils. Collectively, our results demonstrate that high-resolution structure of coiled-coils, as well as parallel and antiparallel orientations of dimers and tetramers, can be accurately predicted from sequence. De novo modeling was also used to generate models of competing oligomeric states, which were used to compare stabilities and thus predict the native stoichiometry from sequence. In a benchmark set of 33 coiled-coil sequences, forming dimers to pentamers, up to 70% of the oligomeric states could be correctly predicted. The calculations demonstrated that the free energy of helix folding could be an important factor for determining stability and oligomeric state of homomeric coiled-coils. The computational methods developed here should be broadly applicable to studies of sequence-structure relationships in coiled-coils and the design of higher order assemblies with improved oligomerization specificity.

A set of computationally designed orthogonal antiparallel homodimers that expands the synthetic coiled-coil toolkit.

Molecular engineering of protein assemblies, including the fabrication of nanostructures and synthetic signaling pathways, relies on the availability of modular parts that can be combined to give different structures and functions. Currently, a limited number of well-characterized protein interaction components are available. Coiled-coil interaction modules have been demonstrated to be useful for biomolecular design, and many parallel homodimers and heterodimers are available in the coiled-coil toolkit. In this work, we sought to design a set of orthogonal antiparallel homodimeric coiled coils using a computational approach. There are very few antiparallel homodimers described in the literature, and none have been measured for cross-reactivity. We tested the ability of the distance-dependent statistical potential DFIRE to predict orientation preferences for coiled-coil dimers of known structure. The DFIRE model was then combined with the CLASSY multistate protein design framework to engineer sets of three orthogonal antiparallel homodimeric coiled coils. Experimental measurements confirmed the successful design of three peptides that preferentially formed antiparallel homodimers that, furthermore, did not interact with one additional previously reported antiparallel homodimer. Two designed peptides that formed higher-order structures suggest how future design protocols could be improved. The successful designs represent a significant expansion of the existing protein-interaction toolbox for molecular engineers.

Transforming Growth Factor (TGF)-β-activated Kinase 1 (TAK1) Activation Requires Phosphorylation of Serine 412 by Protein Kinase A Catalytic Subunit α (PKACα) and X-linked Protein Kinase (PRKX)

TGF-β-activated kinase 1 (TAK1) is a key kinase in mediating Toll-like receptors (TLRs) and interleukin-1 receptor (IL-1R) signaling. Although TAK1 activation involves the phosphorylation of Thr-184 and Thr-187 residues at the activation loop, the molecular mechanism underlying the complete activation of TAK1 remains elusive. In this work, we show that the Thr-187 phosphorylation of TAK1 is regulated by its C-terminal coiled-coil domain-mediated dimerization in an autophosphorylation manner. Importantly, we find that TAK1 activation in mediating downstream signaling requires an additional phosphorylation at Ser-412, which is critical for TAK1 response to proinflammatory stimuli, such as TNF-α, LPS, and IL-1β. In vitro kinase and shRNA-based knockdown assays reveal that TAK1 Ser-412 phosphorylation is regulated by cAMP-dependent protein kinase catalytic subunit α (PKACα) and X-linked protein kinase (PRKX), which is essential for proper signaling and proinflammatory cytokine induction by TLR/IL-1R activation. Morpholino-based in vivo knockdown and rescue studies show that the corresponding site Ser-391 in zebrafish TAK1 plays a conserved role in NF-κB activation. Collectively, our data unravel a previously unknown mechanism involving TAK1 phosphorylation mediated by PKACα and PRKX that contributes to innate immune signaling.

Increasing the affinity of selective bZIP-binding peptides through surface residue redesign.

The coiled-coil dimer is a prevalent protein interaction motif that is important for many cellular processes. The basic leucine-zipper (bZIP) transcription factors are one family of proteins for which coiled-coil mediated dimerization is essential for function, and misregulation of bZIPs can lead to disease states including cancer. This makes coiled coils attractive protein-protein interaction targets to disrupt using engineered molecules. Previous work designing peptides to compete with native coiled-coil interactions focused primarily on designing the core residues of the interface to achieve affinity and specificity. However, folding studies on the model bZIP GCN4 show that coiled-coil surface residues also contribute to binding affinity. Here we extend a prior study in which peptides were designed to bind tightly and specifically to representative members of each of 20 human bZIP families. These "anti-bZIP" peptides were designed with an emphasis on target-binding specificity, with contributions to design-target specificity and affinity engineered considering only the coiled-coil core residues. High-throughput testing using peptide arrays indicated many successes. We have now measured the binding affinities and specificities of anti-bZIPs that bind to FOS, XBP1, ATF6, and CREBZF in solution and tested whether redesigning the surface residues can increase design-target affinity. Incorporating residues that favor helix formation into the designs increased binding affinities in all cases, providing low-nanomolar binders of each target. However, changes in surface electrostatic interactions sometimes changed the binding specificity of the designed peptides.

Stretching desmin filaments with receding meniscus reveals large axial tensile strength.

Desmin forms the intermediate filament system of muscle cells where it plays important role in maintaining mechanical integrity and elasticity. Although the importance of intermediate-filament elasticity in cellular mechanics is being increasingly recognized, the molecular basis of desmin's elasticity is not fully understood. We explored desmin elasticity by molecular combing with forces calculated to be as large as 4nN. Average filament contour length increased 1.55-fold axial on average. Molecular combing together with EGTA-treatment caused the fragmentation of the filament into short, 60 to 120-nm-long and 4-nm-wide structures. The fragments display a surface periodicity of 38nm, suggesting that they are composed of laterally attached desmin dimers. The axis of the fragments may deviate significantly from that of the overstretched filament, indicating that they have a large orientational freedom in spite of being axially interconnected. The emergence of protofibril fragments thus suggests that the interconnecting head or tail domains of coiled-coil desmin dimers are load-bearing elements during axial stretch.

Myosin Storage Myopathy Mutations Disrupt Myofibrillar Assembly/ Stability and Cause Progressive Muscle Degeneration in a Drosophila Model

Myosin storage myopathy (MSM) is a congenital disorder caused by dominant missense mutations in the β-cardiac MHC rod and characterized by subsarcolemmal accumulation of β-cardiac myosin that has a hyaline appearance. These mutations map near to or within the assembly competence domain known to be crucial to filament assembly. The mutations disrupt hydropathy or charge of residues in the heptad repeat, altering interactions that stabilize myosin coiled-coil dimers and thick filaments. This potentially disrupts ordered myofibrillar assembly, causing myofibrillar disarray and myosin aggregation. Our Drosophila models for MSM make it possible to examine interactions between wild-type and mutant full-length myosins for pursuing mechanistic investigations. We introduced the R1845W, L1793P or the E1883K mutation into a Drosophila MHC transgene and expressed each in the indirect flight/jump muscles and in the heart. Our studies show a severe reduction in the flight and jump ability of the transgenic flies in both homozygous and heterozygous states, with an age-dependent worsening of muscle function. Electron and confocal microscopy of the indirect flight muscles of transgenic lines show myofibrillar disarray with large areas of granular/ filamentous inclusions similar to hyaline bodies found in affected humans. Semi-automated optical heartbeat analysis of the mutant heterozygotes shows restrictive cardiac physiology and diastolic dysfunction with evidence of worsening cardiac phenotype with age. Lifespans of the MSM mutants are also reduced in comparison to the transgenic control. Future studies will aim at analyzing in vitro filament forming ability of the mutant myosin to determine if defective filament formation and/or instability of the myosin filaments are the basis of MSM. Our model would also potentially help discern if specific chaperones, small molecule chaperone inducers or enhanced autophagy can ameliorate myopathic defects in MSM.

Intermediate Filament Structure, Assembly and Nanomechanics

Intermediate filaments (IFs) consist of two-stranded coiled coils that form anti-parallel, half-staggered tetramers. By time-lapse electron microscopy, complemented with total internal reflection fluorescence (TIRF) microscopy, we have investigated the in vitro assembly of vimentin to define the assembly pathway for vertebrate cytoplasmic IFs. First, we have characterized the physical and structural state of the soluble vimentin subunits by analytical ultracentrifugation (AUC) and X-ray crystallography. Assembly is induced by a change in the ionic strength and starts with the lateral association of tetramers to full-width unit-length filaments (ULFs) driven by the interaction of the basic, non-structured head domains with the acidic coiled-coil rods. In a next step, ULFs longitudinally anneal by an end-on-addition mechanism to yield filaments. This mechanism is also exhibited by muscle desmin and the epithelial keratins, whereas the nuclear IF proteins, i.e. the lamins, do not assemble into ULFs. In a next step, the subunit composition of ULFs and IFs of different IF proteins was analyzed by scanning transmission electron microscopy (STEM) and cryo-electron tomography of native specimens. Depending on the ionic conditions used for assembly, on average keratin IFs harbor 8, vimentin IFs 16 and desmin IFs 24 coiled-coil dimers per filament cross-section. The formation of ULFs was investigated further by small-angle X-ray scattering (SAXS) and AUC, employing a mutant vimentin variant that is arrested in the ULF state. With these data at hand, we investigated the impact of human disease mutations found in desmin that cause myofibrillar myopathy. Last but not least, we explored the network formation of lamin A and some of its disease variants, which strongly deviates from that of cytoplasmic IFs. These data give a first mechanistic clue how the lamin network provides mechanical stability to the nuclear envelope and nuclear architecture.

Structure of a highly conserved domain of Rock1 required for Shroom-mediated regulation of cell morphology.

Rho-associated coiled coil containing protein kinase (Rho-kinase or Rock) is a well-defined determinant of actin organization and dynamics in most animal cells characterized to date. One of the primary effectors of Rock is non-muscle myosin II. Activation of Rock results in increased contractility of myosin II and subsequent changes in actin architecture and cell morphology. The regulation of Rock is thought to occur via autoinhibition of the kinase domain via intramolecular interactions between the N-terminus and the C-terminus of the kinase. This autoinhibited state can be relieved via proteolytic cleavage, binding of lipids to a Pleckstrin Homology domain near the C-terminus, or binding of GTP-bound RhoA to the central coiled-coil region of Rock. Recent work has identified the Shroom family of proteins as an additional regulator of Rock either at the level of cellular distribution or catalytic activity or both. The Shroom-Rock complex is conserved in most animals and is essential for the formation of the neural tube, eye, and gut in vertebrates. To address the mechanism by which Shroom and Rock interact, we have solved the structure of the coiled-coil region of Rock that binds to Shroom proteins. Consistent with other observations, the Shroom binding domain is a parallel coiled-coil dimer. Using biochemical approaches, we have identified a large patch of residues that contribute to Shrm binding. Their orientation suggests that there may be two independent Shrm binding sites on opposing faces of the coiled-coil region of Rock. Finally, we show that the binding surface is essential for Rock colocalization with Shroom and for Shroom-mediated changes in cell morphology.

Coiled-coil dimerization of the LOV2 domain of the blue-light photoreceptor phototropin 1 fromArabidopsis thaliana

A key role in signal transduction and dimerization mediated by Per-Arnt-Sim (PAS) domains is played by α-helical linkers that flank the structurally similar α/β cores of these domains. However, crystal-packing forces and the different construct lengths and sequences of the PAS domains influence the final length and orientation of the linkers relative to the core and create uncertainty in the exact mechanism of the linker function. Thus, structural characterization and comparison of the linkers within isolated PAS-domain constructs and/or full-length PAS-containing proteins is important for clarification of the mechanism. The plant blue-light photoreceptors phototropins possess two N-terminal flavin mononucleotide-based light, oxygen or voltage (LOV) domains (LOV1 and LOV2) that comprise a subclass of the PAS family and one C-terminal serine/threonine kinase domain whose enzymatic activity is regulated by blue light. The dark-adapted state crystal structures of the Arabidopsis thaliana phototropin 1 and phototropin 2 LOV1-domain constructs flanked by an N-terminal A'α helix and the structure of the phototropin 2 core LOV2 domain are known. Here, the crystal structure of the A. thaliana phototropin 1 LOV2 domain has been determined in its dark-adapted state. The core is flanked by an N-terminal A'α helix and a C-terminal Jα helix similar to those in the previously reported structure of Avena sativa phototropin 1 LOV2. In contrast to the monomeric A.sativa LOV2, A.thaliana LOV2 is a dimer in which two A'α helices adopt a scissor-like orientation at the dimer interface and form a short α-helical coiled coil. The Jα helix predominantly interacts with the β-sheet and plays a role in coiled-coil formation and dimerization.

The Structural Motifs for Substrate Binding and Dimerization of the α Subunit of Collagen Prolyl 4-Hydroxylase

Collagen prolyl 4-hydroxylase (C-P4H) catalyzes the proline hydroxylation of procollagen, an essential modification in the maturation of collagens. C-P4H consists of two catalytic α subunits and two protein disulfide isomerase β subunits. The assembly of these subunits is unknown. The α subunit contains an N domain (1-143), a peptide-substrate-binding-domain (PSB, 144-244) and a catalytic domain (245-517). Here, we report the dimeric structure of the N-terminal region (1-244) of the α subunit. It is shown that the N domain has an important role in the assembly of the C-P4H tetramer, by forming an extended four-helix bundle that includes an antiparallel coiled-coil dimerization motif between the two α subunits. Complexes of this construct with a C-P4H inhibitor and substrate show the mode of peptide-binding to the PSB domain. Both peptides adopt a poly-(L)-proline-type-II helix conformation and bind in a curved, asymmetric groove lined by conserved tyrosines and an Arg-Asp salt bridge.

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].

Design of a single-chain polypeptide tetrahedron assembled from coiled-coil segments

Protein structures evolved through a complex interplay of cooperative interactions and it is still very challenging to design new protein folds de novo. Here, we present a strategy to design self-assembling polypeptide nanostructured polyhedra, based on modularization using orthogonal dimerizing segments. We designed end experimentally demonstrated formation of the tetrahedron that self-assembles from a single polypeptide chain comprising 12 concatenated coiled-coil-forming segments separated by flexible peptide hinges. Path of the polypeptide chain is guided by the defined order of segments that traverse each of the 6 edges of the tetrahedron exactly twice, forming coiled-coil dimers with their corresponding partners. Coincidence of the polypeptide termini in the same vertex is demonstrated by reconstitution of the split fluorescent protein by the polypeptide with the correct tetrahedral topology, while polypeptides with a deleted or scrambled segment order fail to self-assemble correctly. This design platform provides the basis for construction of new topological polypeptide folds based on the set of orthogonal interacting polypeptide segments.

Abstract 790: An alternative tetramerization domain of p53 for exclusive homo-oligomerization and potent tumor suppression.

Abstract The tumor suppressor p53 is mutated in more than 50% of all cancers. Our data have shown that swapping the tetramerization domain (TD) of p53 with an alternative oligomerization domain enhances the utility of p53 for cancer gene therapy. Previously, we have swapped the tetramerization domain of p53 with the coiled-coil (CC) domain from breakpoint cluster region (Bcr). This alteration of the oligomerization motif of the tumor suppressor allowed for our construct, namely p53-CC, to evade hetero-oligomerization with endogenous mutant p53 commonly found in cancer cells. This proves to be critical since mutant p53 has a transdominant inhibitory effect over wild-type p53 upon hetero-oligomerization. A co-immunopreciptation (co-IP) experiment in human breast ductal carcinoma (T47D) cells validated our hypothesis that endogenous p53 interacts directly with exogenous wild-type p53, which is due to hetero-oligomerization via their TDs. In contrast, p53-CC, which lacks the TD, evaded binding to endogenous p53. Furthermore, overexpression of a potent mutant p53 (contains three hotspot mutations; R175H, R248W, and R273H) significantly impaired the function of exogenous wild-type p53, while the tumor suppressor activity of p53-CC was not affected. To further increase the apoptotic potential of p53-CC, rational design of mutations in the CC domain were investigated for two purposes: first to increase the dimerization affinity of CC, and second to prevent any potential interaction with endogenous Bcr. Leucine at residue 62 of the CC domain forms a critical hydrophobic pocket at the dimeric interface. Therefore, mutating this residue into the more hydrophobic isoleucine (with similar molecular weight),, should increase CC dimerization affinity. Results from a standard mammalian two-hybrid assay will be presented to validate the binding affinity of our mutant CC compared to the native CC. Furthermore, two additional mutations on the CC domain (R55E and E34K) were carried out to prevent any potential interaction of our construct with endogenous Bcr. These two mutations introduce a salt bridge that promotes homo-dimerization of mutant CC as well as preventing hetero-dimerization with native CC representative of endogenous Bcr. This will be confirmed using another co-IP experiment that shows no interaction between mutant CC and endogenous Bcr. Finally, two apoptotic assays were carried out to verify that introducing the aforementioned mutations did not cause any loss of tumor suppressor activity of our enhanced p53-CC variant. In summary, a p53 with altered TD has been designed for exclusive homo-dimerization and has potent activity as a tumor suppressor. Our ultimate goal is to use this version of p53 as a new gene therapeutic, capable of bypassing the dominant negative effect in cancers with mutated or mislocalized p53. Citation Format: Abood Okal, Mohanad Mossalam, Karina J. Matissek, Carol S. Lim. An alternative tetramerization domain of p53 for exclusive homo-oligomerization and potent tumor suppression. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 790. doi:10.1158/1538-7445.AM2013-790

Identification and Cytoprotective Function of a Novel Nestin Isoform, Nes-S, in Dorsal Root Ganglia Neurons

In this study, the first nestin isoform, Nes-S, was identified in neurons of dorsal root ganglia (DRG) of adult rats. Nes-S cannot form filaments by itself in cytoplasmic intermediate filament-free SW13 cells. Instead, it co-assembles into filaments with vimentin when transfected into vimentin(+) SW13 cells, and with peripherin and neurofilament proteins when transfected into N2a cells. In primary DRG neurons, endogenous Nes-S co-assembles with peripherin and neurofilament proteins. The expression of Nes-S first appears in DRG at postnatal day 5 and persists to adulthood. Among the adult tissues we examined, the expression of Nes-S is restricted to the sensory and motor neurons. Finally, exogenous Nes-S enhances viability when transfected into N2a cells, and knockdown of endogenous Nes-S impairs the survival of DRG neurons in primary cultures. Taken together, Nes-S is a new neuronal intermediate filament protein that exerts a cytoprotective function in mature sensory and motor neurons.

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.