Coiled-coil Research Articles

Abstract 3817: Recombinant stapled proteins for the treatment of chronic myeloid leukemia

Abstract The oncoprotein Bcr-Abl is the cause of chronic myeloid leukemia (CML). Current therapies target the tyrosine kinase domain of Bcr-Abl, but due to their non-curative nature, resistance to these drugs is common. Therefore, new treatments are needed for those patients refractive to available therapies. Bcr-Abl homo-oligomerization via its N-terminal coiled coil (CC) domain is required for tyrosine kinase activity. Our previous work has shown that it is possible to inhibit Bcr-Abl activity by targeting the CC domain with a peptidomemetic known as CCmut, delivered as a protein using a cell-penetrating peptide (CPP-CCmut). CPP-CCmut selectively enters leukemic cells and inhibits Bcr-Abl, as seen by Western blot, 7AAD, Annexin V, cell proliferation, and colony forming assays. This leukemia cell-permeable CPP-CCmut protein domain is our lead compound for CML therapy, but to be translatable to the clinic it must be modified to improve proteolytic stability. For this reason, we created stapled versions of this CPP-CCmut. Stapled peptides are resistant to proteolysis, and are emerging as a viable strategy for disrupting protein-protein interactions in diseases including HIV and cancer. Until recently, peptide stapling required incorporation of unnatural amino acids; therefore these peptides were created via solid-state synthesis. However, the synthesis of the 81 amino acid CPP-CCmut is no trivial matter. The ability to staple a longer sequence such as our protein domain is now possible using thiol-ene coupling. This exciting new chemistry allows for the stapling of recombinant proteins at substituted cysteine residues (instead of stapling substituted unnatural amino acids incorporated into synthetically made peptides). Thiol-ene coupling is used here to staple recombinant CPP-CCmut, and provides a path for translation of our lead compound to the clinic. Molecular modeling has identified optimal locations for staples on CPP-CCmut, which include 29/36, 36/43, 50/57, 57/64, as well as double staples 29/36-50/57 and 36/43-50/57. These staple locations still allow for dimerization of CPP-CCmut with Bcr-Abl, as indicated by free energy calculations and helicity. The staple locations have also been optimized for maximal coverage from proteolytic degradation. This work acts as a proof-of-concept that proteins (not just peptides) can be stapled, thus greatly expanding the variety and complexity of stapled proteins for use in the exciting field of targeting protein-protein interfaces to treat disease. Citation Format: Benjamin J. Bruno, Sean P. Cornillie, Thomas E. Cheatham, Daniel H. Chou, Carol S. Lim. Recombinant stapled proteins for the treatment of chronic myeloid leukemia. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3817.

Identification of a region in the coiled-coil domain of Smc3 that is essential for cohesin activity

The cohesin complex plays an important role in sister chromatin cohesion. Cohesin's core is composed of two structural maintenance of chromosome (SMC) proteins, called Smc1 and Smc3. SMC proteins are built from a globular hinge domain, a rod-shaped domain composed of long anti-parallel coiled-coil (CC), and a second globular adenosine triphosphatase domain called the head. The functions of both head and hinge domains have been studied extensively, yet the function of the CC region remains elusive. We identified a mutation in the CC of smc3 (L217P) that disrupts the function of the protein. Cells carrying the smc3-L217P allele have a strong cohesion defect and complexes containing smc3-L217P are not loaded onto the chromosomes. However, the mutation does not affect inter-protein interactions in either the core complex or with the Scc2 loader. We show by molecular dynamics and biochemistry that wild-type Smc3 can adopt distinct conformations, and that adenosine triphosphate (ATP) induces the conformational change. The L217P mutation restricts the ability of the mutated protein to switch between the conformations. We suggest that the function of the CC is to transfer ATP binding/hydrolysis signals between the head and the hinge domains. The results provide a new insight into the mechanism of cohesin activity.

Molecular phylogeny and dynamic evolution of disease resistance genes in the legume family.

BackgroundLegumes are the second-most important crop family in agriculture for its economic and nutritional values. Disease resistance (R-) genes play an important role in responding to pathogen infections in plants. To further increase the yield of legume crops, we need a comprehensive understanding of the evolution of R-genes in the legume family.ResultsIn this study, we developed a robust pipeline and identified a total of 4,217 R-genes in the genomes of seven sequenced legume species. A dramatic diversity of R-genes with structural variances indicated a rapid birth-and-death rate during the R-gene evolution in legumes. The number of R-genes transiently expanded and then quickly contracted after whole-genome duplications, which meant that R-genes were sensitive to subsequent diploidization. R proteins with the Coiled-coil (CC) domain are more conserved than others in legumes. Meanwhile, other types of legume R proteins with only one or two typical domains were subjected to higher rates of loss during evolution. Although R-genes evolved quickly in legumes, they tended to undergo purifying selection instead of positive selection during evolution. In addition, domestication events in some legume species preferentially selected for the genes directly involved in the plant-pathogen interaction pathway while suppressing those R-genes with low occurrence rates.ConclusionsOur results provide insights into the dynamic evolution of R-genes in the legume family, which will be valuable for facilitating genetic improvements in the disease resistance of legume cultivars.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2736-9) contains supplementary material, which is available to authorized users.

Structural characterization of the C-terminal coiled-coil domains of wild-type and kidney disease-associated mutants of apolipoprotein L1.

Trypanosomes that cause sleeping sickness endocytose apolipoprotein L1 (APOL1)-containing trypanolytic factors from human serum, leading to trypanolytic death through generation of APOL1-associated lytic pores in trypanosomal membranes. The trypanosome Trypanosoma brucei rhodesiense counteracts trypanolysis by expressing the surface protein serum response-associated (SRA), which can bind APOL1 common variant G0 to block its trypanolytic activity. However, two missense variants in the C terminal predicted coiled-coil (CC) domains of human APOL1 G1 (S342G/I384M) and G2 (ΔN388Y389) decrease or abrogate APOL1 binding to T. brucei rhodesiense SRA, thus preserving APOL1 trypanolytic activity. These evolutionarily selected APOL1 missense variants, found at a high frequency in some populations of African descent, also confer elevated risk of kidney disease. Understanding the SRA-APOL1 interaction and the role of APOL1 G1 and G2 variants in kidney disease demands structural characterization of the APOL1 CC domain. Using CD, heteronuclear NMR, and molecular dynamics (MD) simulation on structural homology models, we report here unique and dynamic solution conformations of nephropathy variants G1 and G2 as compared with the common variant G0. Conformational plasticity in G1 and G2 CC domains led to interhelical α1-α2 approximation coupled with secondary structural changes and delimited motional properties absent in the G0 CC domain. The G1 substitutions conferred local structural changes principally along helix α1, whereas the G2 deletion altered the structure of both helix α2 and helix α1. These dynamic features of APOL1 CC variants likely reflect their intrinsic structural properties, and should help interpret future APOL1 structural studies and define the contribution of APOL1 risk variants to kidney disease.

A Novel Alternative Exon of the Caenorhabditis Elegans Lev‐11 Tropomyosin Gene is used to Express a Head‐Muscle‐Specific Isoform

Tropomyosin (TM) is a coiled‐coil dimer that binds along actin filaments in both muscle and non‐muscle cells. TM is involved in cellular processes including muscle contraction, cell motility, and cell division. In mammals, more than 40 isoforms of TM are expressed from four genes through extensive alternative splicing. Isoform‐specific actin cytoskeletal regulation is also achieved by tissue‐specific expression and subcellular localization. We utilized the nematode Caenorhabditis elegans to study functional diversity of TM isoforms. In C. elegans, previous studies reported four TM isoforms from a single gene, lev‐11, by alternative promotors and splicing. Two high‐molecular‐weight (HMW) isoforms are expressed by use of the upstream promoter and encoded by nine exons; whereas two low‐molecular‐weight isoforms are expressed from the downstream promoter and encoded by seven exons. In these four isoforms, exons 6, 7, and 8 were constitutive. Through analysis of Expressed Sequence Tag sequences and RT‐PCR, we identified a novel exon (Exon 7a) that is alternative to Exon 7b (previously reported as exon 7) and cloned a full‐length cDNA encoding a third HMW isoform containing Exon 7a, which is termed LEV‐11O. We utilized fluorescent reporter mini‐genes and found favored inclusion of exon 7b in the body‐wall muscles of the main body as well as pharyngeal muscles. Exon 7a was alternatively spliced in the body‐wall muscles in the head, with slight overlap where splice variants containing either exon 7a or 7b were both detected. In striated muscles, TM regulates muscle contractility together with the troponin complex. The inhibitory region of troponin I (TNI) is sufficient to stabilize TM in an inhibitory state for actomyosin interaction. Interestingly, although TNI expresses four isoforms from four genes, LEV‐11O and TNI‐3 are both head‐specific isoforms. Mutations in lev‐11 confer resistance to levamisole, an acetylcholine receptor agonist that induces hyper‐contracted muscle paralysis. We examined levamisole sensitivity in nematode strains with a mutation in either lev‐11 exon 7a or 7b, both resulting in amino acid charge reversals. In the presence of levamisole, wild‐type nematodes exhibited muscle hyper‐contraction in the entire body. An amino acid change from within exon 7b (E234K in LEV‐11A) prevented levamisole‐induced hyper‐contraction in the main body but not the head, whereas an amino acid change in exon 7a (E196K in LEV‐11O) prevented levamisole‐induced hyper‐contraction in the head but not in the main body. Sarcomere localization of LEV‐11 proteins was similar in the head and main body and these mutations did not alter localization patterns. These data indicate that exons 7a and 7b have non‐redundant functions for muscle regulation in the head or main body, respectively. Bacterially‐expressed LEV‐11A and LEV‐11O bound actin with similar affinity and mediated inhibition of actomyosin ATPase by a TNI inhibitory peptide. LEV‐11A(E234K) had similar biochemical properties to wild‐type LEV‐11A; however, LEV‐11O(E196K) strongly inhibited actomyosin ATPase even in the absence of the TNI inhibitory peptide. This inhibitory activity was still slightly‐enhanced with the inhibitory peptide, suggesting that enhanced inhibitory effects on actomyosin ATPase is the basis of levamisole resistance. These results suggest that C. elegans utilizes muscle‐type‐specific alternative splicing to produce functionally distinct TM isoforms.Support or Funding InformationNIH 2R01AR048615‐11A1

MiR-335 Regulate Cell Proliferation by Targeting Rho Associated Coiled-coil Forming Protein Kinase 1 in Ovarian Cancer Cells SKOV3

miR-335 Regulate Cell Proliferation by Targeting Rho Associated Coiled-coil Forming Protein Kinase 1 in Ovarian Cancer Cells SKOV3

The Chloroplastic Protein THF1 Interacts with the Coiled-Coil Domain of the Disease Resistance Protein N' and Regulates Light-Dependent Cell Death.

One branch of plant immunity is mediated through nucleotide-binding/Leu-rich repeat (NB-LRR) family proteins that recognize specific effectors encoded by pathogens. Members of the I2-like family constitute a well-conserved subgroup of NB-LRRs from Solanaceae possessing a coiled-coil (CC) domain at their N termini. We show here that the CC domains of several I2-like proteins are able to induce a hypersensitive response (HR), a form of programmed cell death associated with disease resistance. Using yeast two-hybrid screens, we identified the chloroplastic protein Thylakoid Formation1 (THF1) as an interacting partner for several I2-like CC domains. Co-immunoprecipitations and bimolecular fluorescence complementation assays confirmed that THF1 and I2-like CC domains interact in planta and that these interactions take place in the cytosol. Several HR-inducing I2-like CC domains have a negative effect on the accumulation of THF1, suggesting that the latter is destabilized by active CC domains. To confirm this model, we investigated N', which recognizes the coat protein of most Tobamoviruses, as a prototypical member of the I2-like family. Transient expression and gene silencing data indicated that THF1 functions as a negative regulator of cell death and that activation of full-length N' results in the destabilization of THF1. Consistent with the known function of THF1 in maintaining chloroplast homeostasis, we show that the HR induced by N' is light-dependent. Together, our results define, to our knowledge, novel molecular mechanisms linking light and chloroplasts to the induction of cell death by a subgroup of NB-LRR proteins.

Coiled-Coil Probes Identified the Unfolding Pathway of Yeast Phosphoglycerate Kinase

Large multi-domain proteins which have complicated reacting interfaces and dominate the proteomes of life remain mysterious regarding to their folding behaviors and mechanisms. Comprehensive studies towards the understanding of the fundamentals of protein folding mechanisms tend to bias small single-domain monomeric proteins, possibly due to their simplicity and reversible refolding capabilities. Yet the understanding of protein folding behaviors acquired from studying small single-domain proteins are not adequate to generate an accurate extrapolation for predicting the folding mechanisms of proteins that contain more than one domain. Here we present the characterization and interpretation of the unfolding pathway of a model large multi-domain protein yeast phosphoglycerate kinase (PGK) using single-molecule force spectroscopy (SMFS) and insertion of the antiparallel coiled-coil (CC) polypeptide probe we developed previously. When placed into different loop regions inside of yeast PGK, the CC probe affected different portion of the unfolding profile. Statistical comparison between the force-extension (FE) curves of multiple CC inserted yeast PGK's and the wildtype yeast PGK provided insights into figuring out the effects of conformational changes in domains and across the interfaces between domains on the unfolding pathway of the entire yeast PGK protein. Together with coarse-grain simulation modeling of the unfolding pathway, the sequence of the unfolding events occurred along the unfolding pathway of yeast PGK is precisely determined.

Network of protein interactions within the Drosophila inner kinetochore.

The kinetochore provides a physical connection between microtubules and the centromeric regions of chromosomes that is critical for their equitable segregation. The trimeric Mis12 sub-complex of the Drosophila kinetochore binds to the mitotic centromere using CENP-C as a platform. However, knowledge of the precise connections between Mis12 complex components and CENP-C has remained elusive despite the fundamental importance of this part of the cell division machinery. Here, we employ hydrogen–deuterium exchange coupled with mass spectrometry to reveal that Mis12 and Nnf1 form a dimer maintained by interacting coiled-coil (CC) domains within the carboxy-terminal parts of both proteins. Adjacent to these interacting CCs is a carboxy-terminal domain that also interacts with Nsl1. The amino-terminal parts of Mis12 and Nnf1 form a CENP-C-binding surface, which docks the complex and thus the entire kinetochore to mitotic centromeres. Mutational analysis confirms these precise interactions are critical for both structure and function of the complex. Thus, we conclude the organization of the Mis12–Nnf1 dimer confers upon the Mis12 complex a bipolar, elongated structure that is critical for kinetochore function.

Structure of the TBC1D7-TSC1 complex reveals that TBC1D7 stabilizes dimerization of the TSC1 C-terminal coiled coil region.

TSC1 and TSC2 mutations account for the majority of tuberous sclerosis complex cases. The TSC1 and TSC2 proteins assemble into a complex that is stabilized by TBC1D7 through its direct interaction with the TSC1 coiled coil (CC) region. Loss of TBC1D7 is associated with intellectual disability and megalencephaly. Here, we determine the crystal structure of the complex between TBC1D7 and the C-terminal part (residues 939-992) of TSC1-CC. The structure reveals that two TSC1-CCs form a parallel homodimer, which results in the formation of two symmetric surfaces for interaction with TBC1D7. TBC1D7 employs its α4 and α5 helices to interact with the α1 helix of one TSC1 (939-992) molecule mainly through hydrophobic interactions, and simultaneously associates with the other TSC1 (939-992) molecule using the C-terminal tip of its α4 helix. Biochemical and cell biological data demonstrate that TBC1D7 indeed substantially stabilizes the homodimerization of TSC1-CC, and mutations to the critical interface residues greatly compromise this effect. Together, our data reveal the molecular mechanism underlying TBC1D7-mediated stabilization of TSC1 dimerization, and its contribution to the structural integrity of the holo-TSC complex.

The Eucalyptus grandis NBS-LRR Gene Family: Physical Clustering and Expression Hotspots.

Eucalyptus grandis is a commercially important hardwood species and is known to be susceptible to a number of pests and pathogens. Determining mechanisms of defense is therefore a research priority. The published genome for E. grandis has aided the identification of one important class of resistance (R) genes that incorporate nucleotide binding sites and leucine-rich repeat domains (NBS-LRR). Using an iterative search process we identified NBS-LRR gene models within the E. grandis genome. We characterized the gene models and identified their genomic arrangement. The gene expression patterns were examined in E. grandis clones, challenged with a fungal pathogen (Chrysoporthe austroafricana) and insect pest (Leptocybe invasa). One thousand two hundred and fifteen putative NBS-LRR coding sequences were located which aligned into two large classes, Toll or interleukin-1 receptor (TIR) and coiled-coil (CC) based on NB-ARC domains. NBS-LRR gene-rich regions were identified with 76% organized in clusters of three or more genes. A further 272 putative incomplete resistance genes were also identified. We determined that E. grandis has a higher ratio of TIR to CC classed genes compared to other woody plant species as well as a smaller percentage of single NBS-LRR genes. Transcriptome profiles indicated expression hotspots, within physical clusters, including expression of many incomplete genes. The clustering of putative NBS-LRR genes correlates with differential expression responses in resistant and susceptible plants indicating functional relevance for the physical arrangement of this gene family. This analysis of the repertoire and expression of E. grandis putative NBS-LRR genes provides an important resource for the identification of novel and functional R-genes; a key objective for strategies to enhance resilience.

De Novo Design of Xeno-Metallo Coiled Coils.

Bioinorganic chemists aspire to achieve the same exquisite and highly controlled inorganic chemistry featured in biology. An exciting mimetic approach involves the use of miniature artificial protein scaffolds designed de novo (often based on the coiled coil (CC) scaffold), for reproducing native metal ion sites and their function. Recently, there is increased interest, instead, in the design of xeno-metal sites within CC assemblies. This involves incorporating either non-biological metal ions, cofactors or non-proteinogenic amino acid ligands for metal ion coordination, whilst retaining a minimal CC protein scaffold. Using this approach, one should be able to create functional designs with unique and unusual properties, which combine the advantages of both biology and 'traditional' non-biological inorganic chemistry. It is the recent progress with respect to the design of xeno-metallo CCs which will be discussed in this Focus Review.

Frequent Activating Somatic Alterations in T-Cell Receptor / NF-κb Signaling in Adult T-Cell Leukemia/Lymphoma

Adult T-cell leukemia/lymphoma (ATL) is a peripheral T-cell neoplasm of largely unknown genetic basis, which is associated with human T-cell leukemia virus type-1 (HTLV-1) infection. To delineate a genetic landscape of somatic alterations in ATL, we have performed an integrated genetic study, in which whole-genome/exome (WGS/WES) and transcriptome sequencing (RNA-seq) was performed for a cohort of 83 paired ATL samples, followed by extensive validation using targeted sequencing of detected mutations in 370 follow-up samples.A striking feature of driver lesions in ATL was their strong enrichment in the components of T-cell receptor (TCR) / NF-κB pathway. Accounting for more than 90% of ATL cases, these lesions were characterized by the predominance of activating alterations, including hotspot missense mutations in PLCG1 (36%), PRKCB (33%), CARD11 (24%), VAV1 (18%), IRF4 (14%) and FYN (4%). Among these, most frequently mutated was PLCG1, which encodes phospholipase C γ1 (PLCγ1), a key regulator of the proximal TCR signaling. Besides the S345F and S520F mutations recently reported in cutaneous T-cell lymphoma, we identified an additional hotspot mutations (R48W, E1163K, and D1165H).The second most frequently mutated gene was PRKCB, encoding a member of the protein kinase C (PKC) family of proteins (PKCβ), a pivotal signaling molecule downstream of PLCγ. The frequent mutations of PKCβ were unexpected, because it is PKCθ that has been implicated in TCR signaling, whereas PKCβ has been more focused in the context of B-cell receptor signaling. Approximately 93% of the PRKCB mutations were confined to the catalytic domain with a prominent hotspot at D427, suggesting gain-of-function nature of these mutations. Consistent with this, when transduced with the D427N PKCβ mutant, HEK293T and/or Jurkat cells showed increased membrane translocation after PMA/Ionomycin-stimulation, enhanced IKK phosphorylation and p65 nuclear translocation, and augmented NF-κB transcription, compared to wild-type PKCβ-transduced cells. Thus, these PRKCB mutations are the first activating mutations of this family identified in human cancers.Downstream to PKC lies CARD11, a scaffolding protein required for antigen receptor-induced NF-κB activation. Although previously reported in B-cell lymphomas, CARD11 mutations were more common in ATL (24%). In B-cell lymphomas, mutations are largely limited to the coiled-coil (CC) domain, whereas in ATL, they were clustered not only within the CC domain, but also within the PKC-responsive inhibitory domain, showing a prominent mutational hotspot at E626. The inhibitory domain has been implicated in autoinhibition, whose deletion leads to constitutive activation of CARD11. Intriguingly, WGS identified small intragenic deletions confined to this domain (exons 14-17) in 4 cases (8%) without canonical mutations, and RNA-seq confirmed the skipping of the corresponding exons in these cases. Remarkably, CARD11 mutation significantly co-occurred with PRKCBmutations, suggesting potential functional synergism between these lesions. Actually, overexpression of wild-type CARD11 induced NF-κB activation, which was further augmented by E626K mutation. Similarly, when both CARD11 (E626K) and PRKCB (D427N) mutants were co-expressed, more enhanced NF-κB activation was observed.RNA-seq and follow-up RT-PCR screening also identified novel gene fusions in TCR / NF-κB pathway: five CTLA4-CD28 and three ICOS-CD28 fusions were observed in seven (7%) of the 105 cases examined, of whom one patient carried both chimeric fusions. WGS revealed tandem duplications of 2q33.2 segments containing CD28, CTLA4, and ICOS, compatible with the corresponding fusion transcripts. B7/CD28 co-signaling molecules, including CD28, CTLA4, and ICOS co-receptors, play pivotal roles in positive and negative regulations of TCR signaling. All the predicted chimeric proteins had the cytoplasmic part of CD28, and are expected to be expressed under the control of the regulatory element of CTLA4 or ICOS, likely leading to prolonged expression of CD28 co-stimulator.Our findings suggest that deregulated TCR / NF-κB pathway caused by genetic alterations is a hallmark of ATL pathogenesis. The predominance of gain-of-function mutations in this pathway offers good opportunities for exploiting these mutations for the targets of novel drugs to better manage patients. DisclosuresTobinai:Gilead Sciences: Research Funding. Miyazaki:Sumitomo Dainippon: Honoraria; Celgene Japan: Honoraria; Chugai: Honoraria, Research Funding; Shin-bio: Honoraria; Kyowa-Kirin: Honoraria, Research Funding. Watanabe:Daiichi Sankyo Co., Ltd.: Research Funding.

Dominant spinal muscular atrophy is caused by mutations in BICD2, an important golgin protein.

Spinal muscular atrophies (SMAs) are characterized by degeneration of spinal motor neurons and muscle weakness. Autosomal recessive SMA is the most common form and is caused by homozygous deletions/mutations of the SMN1 gene. However, families with dominant inherited SMA have been reported, for most of them the causal gene remains unknown. Recently, we and others have identified heterozygous mutations in BICD2 as causative for autosomal dominant SMA, lower extremity-predominant, 2 (SMALED2) and hereditary spastic paraplegia (HSP). BICD2 encodes the Bicaudal D2 protein, which is considered to be a golgin, due to its coiled-coil (CC) structure and interaction with the small GTPase RAB6A located at the Golgi apparatus. Golgins are resident proteins in the Golgi apparatus and form a matrix that helps to maintain the structure of this organelle. Golgins are also involved in the regulation of vesicle transport. In vitro overexpression experiments and studies of fibroblast cell lines derived from patients, showed fragmentation of the Golgi apparatus. In the current review, we will discuss possible causes for this disruption, and the consequences at cellular level, with a view to better understand the pathomechanism of this disease.

Large-scale transcriptional profiling of lignified tissues in Tectona grandis.

BackgroundCurrently, Tectona grandis is one of the most valuable trees in the world and no transcript dataset related to secondary xylem is available. Considering how important the secondary xylem and sapwood transition from young to mature trees is, little is known about the expression differences between those successional processes and which transcription factors could regulate lignin biosynthesis in this tropical tree. Although MYB transcription factors are one of the largest superfamilies in plants related to secondary metabolism, it has not yet been characterized in teak. These results will open new perspectives for studies of diversity, ecology, breeding and genomic programs aiming to understand deeply the biology of this species.ResultsWe present a widely expressed gene catalog for T. grandis using Illumina technology and the de novo assembly. A total of 462,260 transcripts were obtained, with 1,502 and 931 genes differentially expressed for stem and branch secondary xylem, respectively, during age transition. Analysis of stem and branch secondary xylem indicates substantial similarity in gene ontologies including carbohydrate enzymes, response to stress, protein binding, and allowed us to find transcription factors and heat-shock proteins differentially expressed. TgMYB1 displays a MYB domain and a predicted coiled-coil (CC) domain, while TgMYB2, TgMYB3 and TgMYB4 showed R2R3-MYB domain and grouped with MYBs from several gymnosperms and flowering plants. TgMYB1, TgMYB4 and TgCES presented higher expression in mature secondary xylem, in contrast with TgMYB2, TgHsp1, TgHsp2, TgHsp3, and TgBi whose expression is higher in young lignified tissues. TgMYB3 is expressed at lower level in secondary xylem.ConclusionsExpression patterns of MYB transcription factors and heat-shock proteins in lignified tissues are dissimilar when tree development was evaluated, obtaining more expression of TgMYB1 and TgMYB4 in lignified tissues of 60-year-old trees, and more expression in TgHsp1, TgHsp2, TgHsp3 and TgBi in stem secondary xylem of 12-year-old trees. We are opening a door for further functional characterization by reverse genetics and marker-assisted selection with those genes. Investigation of some of the key regulators of lignin biosynthesis in teak, however, could be a valuable step towards understanding how rigidity of teak wood and extractives content are different from most other woods. The obtained transcriptome data represents new sequences of T. grandis deposited in public databases, representing an unprecedented opportunity to discover several related-genes associated with secondary xylem such as transcription factors and stress-related genes in a tropical tree.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-015-0599-x) contains supplementary material, which is available to authorized users.

The Effect of Mutations in the Inserted Domain of ATP-Dependent Lon Protease from E. coli on the Enzyme Function

ATP-Dependent protease LonA from E. coli (Ec-Lon), belonging to the superfamily of AAA+ proteins, is a key member of the protein quality control system in bacterial cells. Ec-Lon functions as homohexamer and degrades abnormal and defective polypeptides as well as a number of regulatory proteins by the processive mechanism. Ec-Lon subunit includes--the both ATPase and proteolytic components (AAA+ module and P domain) in addition to the unique non-catalytic region formed by the N-terminal (N) and the inserted c-helical (HI(CC)) domains. The mutant forms Lon-R164A, Lon-R192A and Lon-Y294A have been obtained and characterized in order to reveal the role of the HI (CC) domain for the enzyme functioning. C-Terminal part of the HI (CC) domain is shown to display an allosteric effect on the efficiency of the enzyme ATPase and proteolytic sites while its coiled-coil (CC) region is involved in the interaction with the protein substrate.

Cytoplasmic and Nuclear Localizations Are Important for the Hypersensitive Response Conferred by Maize Autoactive Rp1-D21 Protein.

Disease resistance (R) genes have been isolated from many plant species. Most encode nucleotide binding leucine-rich repeat (NLR) proteins that trigger a rapid localized programmed cell death called the hypersensitive response (HR) upon pathogen recognition. Despite their structural similarities, different NLR are distributed in a range of subcellular locations, and analogous domains play diverse functional roles. The autoactive maize NLR gene Rp1-D21 derives from an intragenic recombination between two NLR genes, Rp1-D and Rp1-dp2, and confers a HR independent of the presence of a pathogen. Rp1-D21 and its N-terminal coiled coil (CC) domain (CCD21) confer autoactive HR when transiently expressed in Nicotiana benthamiana. Rp1-D21 was predominantly localized in cytoplasm with a small amount in the nucleus, while CCD21 was localized in both nucleus and cytoplasm. Targeting of Rp1-D21 or CCD21 predominantly to either the nucleus or the cytoplasm abolished HR-inducing activity. Coexpression of Rp1-D21 or CCD21 constructs confined, respectively, to the nucleus and cytoplasm did not rescue full activity, suggesting nucleocytoplasmic movement was important for HR induction. This work emphasizes the diverse structural and subcellular localization requirements for activity found among plant NLR R genes.

CC-NBS-LRR-Type R Proteins for Rice Blast Commonly Interact with Specific WRKY Transcription Factors

Rice blast resistance caused by Resistance (R) genes, most of which encode coiled-coil (CC), nucleotide-binding (NB), and leucine-rich-repeat (LRR) domains, is race-specific. Panicle blast 1 (Pb1) is a panicle blast resistance gene that confers a durable non-race-specific blast resistance to rice. Despite being non-R, Pb1 also encodes a CC-NB-LRR protein. Pb1 specifically interacts with rice WRKY45, a transcription factor (TF) in the rice salicylic acid signaling pathway, through its CC domain, and that this interaction is important for the blast resistance conferred by Pb1. Additionally, barley MLA10, an R protein for Blumeria graminis, specifically interacts with HvWRKY1/2. Here, we investigated whether the blast resistance between conferred by R genes and non-R Pb1 genes is mediated by their interaction with WRKY TFs. We performed targeted yeast two-hybrid and glutathione S-transferase (GST)-pull-down assays, which showed that all the five rice R proteins of the CC-NB-LRR type tested, Pi36, Pib, Pita, Pit, and Piz-t, interacted with WRKY45 and WRKY66 through their CC domains. However, elicitor-triggered immunity due to these R genes was not affected by the knockdown of these WRKY genes. The results suggest that R proteins can potentially transduce defense signals by interacting with WRKY TFs but the potentials are masked by HR-mediated elicitor-triggered immunity responses.

Abstract 2641: Inhibition of Bcr-Abl in human leukemic cells with a coiled coil protein delivered by a leukemia-specific cell penetrating peptide

Abstract The oncoprotein Bcr-Abl is the cause of chronic myeloid leukemia (CML). Current CML therapies target the tyrosine kinase domain of Bcr-Abl, but due to their non-curative nature, resistance to these drugs is common. Therefore, new treatments are needed for those patients refractive to available therapies. Bcr-Abl homo-oligomerization via its N-terminal coiled coil (CC) domain is required for tyrosine kinase activity. Our previous work has shown that it is possible to inhibit Bcr-Abl activity by targeting the CC domain with a altered version of the CC domain, known as CCmut3, delivered as a plasmid. In this study, CCmut3 is delivered to cells as a protein by utilizing a leukemia-specific cell-penetrating peptide (CPP). Here, recombinant CPP-CCmut3 was expressed, purified, and tested for its anti-oncogenic activity. CPP-CCmut3 was able to enter two leukemic cell lines (K562 and Ba/F3-P210) and inhibit Bcr-Abl activity as shown by induction of necrosis/apoptosis via 7-AAD/Annexin V staining, reduction of oncogenic potential in colony forming assays, reduction of cell proliferation, and inhibition of Bcr-Abl phosphorylation (kinase activity). Further, CPP-CCmut3 did not enter non-leukemic cell lines (HEK293 and MCF-7). While CPP-CCmut3 was able to enter the parental, non-leukemic Bcr-Abl- Ba/F3 pro-B cell line, it revealed no signs of activity in the assays performed, as expected. These results indicate the feasibility of using CPP-CCmut3 as a therapeutic against CML. Citation Format: Benjamin J. Bruno, Carol S. Lim. Inhibition of Bcr-Abl in human leukemic cells with a coiled coil protein delivered by a leukemia-specific cell penetrating peptide. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2641. doi:10.1158/1538-7445.AM2015-2641

AB169. CCDC34 is up-regulated in bladder cancer and its silencing suppresses bladder cancer cell proliferation and migration

ObjectiveThe coiled coil is a superhelical structural protein motif that has been thoroughly investigated in recent years and coiled-coil domain-containing proteins have exhibited a large diversity of function in biological systems (e.g., gene regulation, cell division, membrane fusion, drug extrusion). The aim of this study was to investigate the critical role of coiled-coil domain-containing protein 34 (CCDC34) in bladder carcinogenesis, which has never been reported to date.MethodsImmunohistochemical staining and western blot were used to detect CCDC34 expression in bladder cancers specimens and cell lines. Lentivirus-mediated small interfering RNA (siRNA) against CCDC34 was designed and the silencing effect was examined by quantitative RT-PCR in bladder cancer cell lines T24 and 5637. The biological functions of CCDC34 knockdown on bladder cancer cells were investigated by examining cell proliferation using a high content screening (HCS) assay, BrdU incorporation assay and colony formation assay, as well as cell migration by in vitro wound healing assay. Cell cycle distribution and apoptosis were detected by flow cytometric analysis. The expressions of c-Raf and c-Jun as well as the phosphorylation of MEK, ERK1/2, JNK, P38 and AKT were also measured using Western blot. We further investigated the effect of therapeutic siRNA targeting CCDC34 on T24 xenograft tumor growth in nude mice.ResultsCCDC34 level was significantly increased in human bladder cancer tissues and cell lines. CCDC34 knockdown significantly suppressed bladder cancer cells proliferation and migration, and induced cell cycle arrest at G2/M phase and increased apoptosis in vitro. In addition, intratumor delivery of therapeutic siRNA targeting CCDC34 elicited delayed tumor growth of bladder cancer xenograft in nude mice. Moreover, CCDC34 knockdown decreased the phosphorylation of MEK, ERK1/2, JNK, p38 and Akt, and the expressions of c-Raf and c-Jun, indicating MAPK and AKT pathways (ERK/MAPK, p38/MAPK, JNK/MAPK and PI3K/Akt) might be involved in CCDC34 modulation of bladder cancer cell proliferation and migration.ConclusionsOur findings revealed for the first time a potential oncogenic role for CCDC34 in bladder carcinoma pathogenesis and it may serve as a biomarker or even a therapeutic target for bladder cancer.