CR Domain Research Articles

Evolution of Einkorn wheat centromeres is driven by the mutualistic interplay of two LTR retrotransposons

BackgroundCentromere function is highly conserved across eukaryotes, but the underlying centromeric DNA sequences vary dramatically between species. Centromeres often contain a high proportion of repetitive DNA, such as tandem repeats and/or transposable elements (TEs). Einkorn wheat centromeres lack tandem repeat arrays and are instead composed mostly of the two long terminal repeat (LTR) retrotransposon families RLG_Cereba and RLG_Quinta which specifically insert in centromeres. However, it is poorly understood how these two TE families relate to each other and if and how they contribute to centromere function and evolution.ResultsBased on conservation of diagnostic motifs (LTRs, integrase and primer binding site and polypurine-tract), we propose that RLG_Cereba and RLG_Quinta are a pair of autonomous and non-autonomous partners, in which the autonomous RLG_Cereba contributes all the proteins required for transposition, while the non-autonomous RLG_Quinta contributes GAG protein. Phylogenetic analysis of predicted GAG proteins showed that the RLG_Cereba lineage was present for at least 100 million years in monocotyledon plants. In contrast, RLG_Quinta evolved from RLG_Cereba between 28 and 35 million years ago in the common ancestor of oat and wheat. Interestingly, the integrase of RLG_Cereba is fused to a so-called CR-domain, which is hypothesized to guide the integrase to the functional centromere. Indeed, ChIP-seq data and TE population analysis show only the youngest subfamilies of RLG_Cereba and RLG_Quinta are found in the active centromeres. Importantly, the LTRs of RLG_Quinta and RLG_Cereba are strongly associated with the presence of the centromere-specific CENH3 histone variant. We hypothesize that the LTRs of RLG_Cereba and RLG_Quinta contribute to wheat centromere integrity by phasing and/or placing CENH3 nucleosomes, thus favoring their persistence in the competitive centromere-niche.ConclusionOur data show that RLG_Cereba cross-mobilizes the non-autonomous RLG_Quinta retrotransposons. New copies of both families are specifically integrated into functional centromeres presumably through direct binding of the integrase CR domain to CENH3 histone variants. The LTRs of newly inserted RLG_Cereba and RLG_Quinta elements, in turn, recruit and/or phase new CENH3 deposition. This mutualistic interplay between the two TE families and the plant host dynamically maintains wheat centromeres.

The use of cyclic peptide antigens to generate LRP8 specific antibodies

LRP8 is a member of the LDLR-like protein family. It is a transport receptor, which can be used in the design of antibodies specific for investigating increasing exposure to therapeutics with respect to the blood brain barrier (BBB). In this study, a LRP8 peptide immunization strategy was implemented to generate antibodies to a specific epitope of the CR1 domain of LRP8 that could enable transport function and cross-react in mice, cynomolgus monkeys and humans. Additionally, a cyclized peptide immunogen was designed to conserve the structural β-hairpin element observed in a previously solved crystal structure of a related CR domain. As a result of this structure-based antigenic design, an LRP8 specific antibody, 11H1, was selected and characterized in ligand binding assays and crystallographic structure determination. The high-resolution structure of the 11H1 Fab complexed to the cyclized CR1 peptide revealed key interactions driving epitope recognition that were confirmed using a site-directed mutagenesis approach. A critical observation was that the identified structural CR1 epitope of 11H1 did not compete with reelin’s recognition of CR1 allowing for simultaneous binding. This was predicted by an in silico ternary model and confirmed by reelin binding data. These simultaneous binding events (11H1/CR1/reelin) could therefore enable the CR1 domain of LRP8, 11H1 and reelin to be used as a “BBB transporter” ternary complex in the design of therapeutic proteins. More importantly, 11H1 showed enhanced brain penetration after systemic intravenous dosing in a mouse study, which confirmed its potential function as BBB transporter for therapeutic proteins.

Abstract 3088: Identification of insulin-like growth factor 1 receptor as a novel and specific target of heparin hexasaccharide that selectively inhibits cancer stem cells

Abstract Introduction: Earlier, we discovered that heparin hexasaccharide (HS06), but not shorter or longer polymeric glycosaminoglycan (GAGs), selectively inhibit cancer stem cells (CSCs). Insulin-like growth factor-1 (IGF1) receptor (IGF1R) plays a critical role in regulating CSC phenotype. However, unlike many growth factor receptors, GAG and IGF1R interaction and consequences have not been characterized. Methods: Colon cancer HT29 and HCT-116 cells were grown on ornithine as spheroids. Stable IGF1R knockdown cells (KD) were generated using shRNA transfection and selection. Immunofluorescence (IF) studies were performed with fluorescent-tagged HS06, GAG polymer, and pIGF1R antibody. Images were obtained using a Cytation 5 and analyzed using Gen5. Computational and molecular dynamics studies were employed to determine the binding site and nature of the interaction between HS06 and IGF1R. The impact of this interaction on activation and CSC phenotype was determined using standard techniques such as western blotting and Q-PCR. Results: Fluorescent-tagged HS06 and polymeric GAG, both bound to the colon cancer cell membrane (CM) in a dose-dependent fashion. Intriguingly, HS06 but not polymeric GAG's binding to the cell surface was abolished by IGF1R knockdown, suggesting IGF1R as a specific target HS06. Computational studies identified L1 and CR domain in the N-terminus of IGF1R as a binding site for HS06 but not multimeric GAGs. Intriguingly, there is an overlap between HS06 and IGF1 binding sites on IGF1R. Pretreatment with three different IGF1R N-terminus targeting antibodies reversed HS06 binding to CM, suggesting the N-terminus domain as a likely site of HS06 and IGF1R interactions. Additionally, IGF1 pretreatment also reversed the binding of HS06 in a dose-dependent fashion. Functionally, HS06 (but not polymeric GAG) inhibited IGF1R phosphorylation and CSC phenotype (2° spheroids and/or CSC markers). Both these effects were reversed by pretreatment with IGF1 suggesting competitive inhibition of IGF1 induced activation of IGF1R by HS06. Molecular dynamics studies identified key N-terminus residues in the L1 and CR domain of the N-terminus portion of the IGF1R distinct from IGF1 binding residues. Key basic amino acid residues that were predicted to bind specifically to HS06 in MD simulation have been observed to be mutated in cancer, and IF studies in HT29 cells expressing HS06 binding-site mutant proteins are ongoing. Conclusion: We have uncovered a novel, specific and functionally relevant interaction between HS06 and the N-terminal domain of IGF1R. The HS06-IGF1R interaction induces competitive inhibition of IGF1 induced activation of IGF1R and CSC phenotype. The findings significantly impact an understanding of GAG mediated regulation of the CSC phenotype and present a highly novel mechanism of targeting IGF1R to develop novel anti-cancer agents Citation Format: Rio S. Boothello, Jyothi C. Sistla, Nehru Viji Sankaranarayanan. Identification of insulin-like growth factor 1 receptor as a novel and specific target of heparin hexasaccharide that selectively inhibits cancer stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3088.

Ontologies for cloud robotics

AbstractCloud robotics (CR) is currently a growing area in the robotic community. Indeed, the use of cloud computing to share data and resources of distributed robotic systems leads to the design and development of cloud robotic systems (CRS) which constitute useful technologies for a wide range of applications such as smart manufacturing, aid and rescue missions. However, in order to get coherent agent-to-cloud communications and efficient agent-to-agent collaboration within these CRS, there is a need to formalize the knowledge representation in CR. Hence, the use of ontologies provides a mean to define formal concepts and their relations in an interoperable way. This paper presents standard robotic ontologies and their extension in the CR domain as well as their possible implementations in the case of a real-world CR scenario.

Analysis of genetic and clinical characteristics of a Chinese Kallmann syndrome cohort with ANOS1 mutations.

To analyze ANOS1 gene mutations in a large Chinese Kallmann syndrome (KS) cohort and to characterize the clinical presentation of the disease in patients with ANOS1 mutations. Chinese patients with KS, including 187 sporadic and 23 pedigree cases were recruited. Patients' ANOS1 gene sequences were analyzed by direct sequencing of PCR-amplified products. In silico analysis was used to assess functional relevance of newly identified missense mutations. Patients' clinical characteristics were analyzed retrospectively. Fifteen nonsynonymous rare ANOS1 variants were found in 13 out of 187 sporadic and 8 out of 23 familial IHH probands. Seven novel (C86F, C90Y, C151W, Y379X, c.1062 + 1G > A, Y579L fs 591X, R597X) and eight recurrent ANOS1 mutations (S38X, R257X, R262X, R423X, R424X, V560I, c.1843-1G > A, p.R631X) were identified. All the novel mutations were predicted to be pathogenic. The prevalence of cryptorchidism was high (38.1%) and occurred in patients with different kind of ANOS1 mutations, while the patients with the same mutation did not present with cryptorchidism uniformly. The prevalence of ANOS1 gene mutations is low in sporadic KS patients, but is much higher in familial KS patients. In the present study, we identify seven novel ANOS1 mutations, including two mutations in the CR domain, which are probably pathogenic. These mutations expand the ANOS1 mutation spectrum and provide a foundation for prenatal diagnosis and genetic counseling.

The High Affinity Binding Site on Plasminogen Activator Inhibitor-1 (PAI-1) for the Low Density Lipoprotein Receptor-related Protein (LRP1) Is Composed of Four Basic Residues

Plasminogen activator inhibitor 1 (PAI-1) is a serpin inhibitor of the plasminogen activators urokinase-type plasminogen activator (uPA) and tissue plasminogen activator, which binds tightly to the clearance and signaling receptor low density lipoprotein receptor-related protein 1 (LRP1) in both proteinase-complexed and uncomplexed forms. Binding sites for PAI-1 within LRP1 have been localized to CR clusters II and IV. Within cluster II, there is a strong preference for the triple CR domain fragment CR456. Previous mutagenesis studies to identify the binding site on PAI-1 for LRP1 have given conflicting results or implied small binding contributions incompatible with the high affinity PAI-1/LRP1 interaction. Using a highly sensitive solution fluorescence assay, we have examined binding of CR456 to arginine and lysine variants of PAI-1 and definitively identified the binding site as composed of four basic residues, Lys-69, Arg-76, Lys-80, and Lys-88. These are highly conserved among mammalian PAI-1s. Individual mutations result in a 13-800-fold increase in Kd values. We present evidence that binding involves engagement of CR4 by Lys-88, CR5 by Arg-76 and Lys-80, and CR6 by Lys-69, with the strongest interactions to CR5 and CR6. Collectively, the individual binding contributions account quantitatively for the overall PAI-1/LRP1 affinity. We propose that the greater efficiency of PAI-1·uPA complex binding and clearance by LRP1, compared with PAI-1 alone, is due solely to simultaneous binding of the uPA moiety in the complex to its receptor, thereby making binding of the PAI-1 moiety to LRP1 a two-dimensional surface-localized association.

Interaction of Fibrin with the Very Low Density Lipoprotein Receptor: Further Characterization and Localization of the Fibrin-Binding Site.

Our recent study revealed that fibrin interacts with the very low density lipoprotein receptor (VLDLR) on endothelial cells through its βN domains, and this interaction promotes transendothelial migration of leukocytes and thereby inflammation. The major aims of this study were to further characterize this interaction and localize the fibrin-binding site in the VLDLR. To localize the fibrin-binding site, we expressed a soluble extracellular portion of this receptor, sVLDLRHT, its N- and C-terminal regions, VLDLR(1-8)HT and des(1-8)VLDLRHT, respectively, and a number of VLDLR fragments containing various combinations of CR domains and confirmed their proper folding by fluorescence spectroscopy. Interaction of these fragments with the (β15-66)2 fragment corresponding to a pair of VLDLR-binding βN domains of fibrin was tested by different methods. Our experiments performed by an enzyme-linked immunosorbent assay and surface plasmon resonance revealed that the VLDLR(1-8)HT fragment containing eight CR domains of VLDLR and its subfragments, VLDLR(1-4)HT and VLDLR(2-4)HT, interact with (β15-66)2 with practically the same affinity as sVLDLRHT while the affinity of VLDLR(2-3)HT was ∼2-fold lower. In contrast, des(1-8)VLDLRHT exhibited no binding. Formation of the complex in solution between the fibrin-binding fragments of VLDLR and (β15-66)2 was detected by fluorescence spectroscopy. In addition, formation of a complex between VLDLR(2-4)HT and (β15-66)2 in solution was confirmed by size-exclusion chromatography. Thus, the results obtained indicate that minimal fibrin-binding structures are located within the second and third CR domains of the VLDL receptor and the presence of the fourth CR domain is required for high-affinity binding. They also indicate that tryptophan residues of CR domains are involved in this binding.

Contribution of the CR domain to P-selectin lectin domain allostery by regulating the orientation of the EGF domain.

The allostery of P-selectin has been studied extensively with a focus on the Lec and EGF domains, whereas the contribution of the CR domain remains unclear. Here, molecular dynamics simulations (MDS) combined with homology modeling were preformed to investigate the impact of the CR domain on P-selectin allostery. The results indicated that the CR domain plays a role in the allosteric dynamics of P-selectin in two ways. First, the CR1 domain tends to stabilize the low affinity of P-selectin during the equilibration processes with the transition inhibition from the S1 to S1’ state by restraining the extension of the bent EGF orientation, or with the relaxation acceleration of the S2 state by promoting the bending of the extended EGF orientation. Second, the existence of CR domain increases intramolecular extension prior to complex separation, increasing the time available for the allosteric shift during forced dissociation with a prolonged bond duration. These findings further our understanding of the structure-function relationship of P-selectin with the enriched micro-structural bases of the CR domain.

Quantitative Dissection of the Binding Contributions of Ligand Lysines of the Receptor-associated Protein (RAP) to the Low Density Lipoprotein Receptor-related Protein (LRP1)

Although lysines are known to be critical for ligand binding to LDL receptor family receptors, relatively small reductions in affinity have been found when such lysines have been mutated. To resolve this paradox, we have examined the specific binding contributions of four lysines, Lys-253, Lys-256, Lys-270, and Lys-289, in the third domain (D3) of receptor-associated protein (RAP), by eliminating all other lysine residues. Using D3 variants containing lysine subsets, we examined binding to the high affinity fragment CR56 from LRP1. With this simplification, we found that elimination of the lysine pairs Lys-253/Lys-256 and Lys-270/Lys-289 resulted in increases in Kd of 1240- and 100,000-fold, respectively. Each pair contributed additively to overall affinity, with 61% from Lys-270/Lys-289 and 39% from Lys-253/Lys-256. Furthermore, the Lys-270/Lys-289 pair alone could bind different single CR domains with similar affinity. Within the pairs, binding contributions of Lys-270 ≫ Lys-256 > Lys-253 ∼ Lys-289 were deduced. Importantly, however, Lys-289 could significantly compensate for the loss of Lys-270, thus explaining how previous studies have underestimated the importance of Lys-270. Calorimetry showed that favorable enthalpy, from Lys-256 and Lys-270, overwhelmingly drives binding, offset by unfavorable entropy. Our findings support a mode of ligand binding in which a proximal pair of lysines engages the negatively charged pocket of a CR domain, with two such pairs of interactions (requiring two CR domains), appropriately separated, being alone sufficient to provide the low nanomolar affinity found for most protein ligands of LDL receptor family members.

Evolution of extracellular Dpp modulators in insects: The roles of tolloid and twisted-gastrulation in dorsoventral patterning of the Tribolium embryo

The formation of the BMP gradient which patterns the DV axis in flies and vertebrates requires several extracellular modulators like the inhibitory protein Sog/Chordin, the metalloprotease Tolloid (Tld), which cleaves Sog/Chordin, and the CR domain protein Twisted gastrulation (Tsg). While flies and vertebrates have only one sog/ chordin gene they possess several paralogues of tld and tsg. A simpler and probably ancestral situation is observed in the short-germ beetle Tribolium castaneum ( Tc), which possesses only one tld and one tsg gene. Here we show that in T. castaneum tld is required for early BMP signalling except in the head region and Tc-tld function is, as expected, dependent on Tc-sog. In contrast, Tc-tsg is required for all aspects of early BMP signalling and acts in a Tc-sog-independent manner. For comparison with Drosophila melanogaster we constructed fly embryos lacking all early Tsg activity (tsg;;srw double mutants) and show that they still establish a BMP signalling gradient. Thus, our results suggest that the role of Tsg proteins for BMP gradient formation has changed during insect evolution.

Identification of NanE as the Thioesterase for Polyether Chain Release in Nanchangmycin Biosynthesis

The polyketide synthase (PKS) for the biosynthesis of the polyether nanchangmycin lacks an apparent thioesterase comparable to the type I thioesterase domains of the modular PKSs responsible for macrolide biosynthesis. Three candidate polyether chain-releasing factors were examined. Both the putative CR domain and the NanE protein appeared to be genetically relevant. Among the three heterologously expressed soluble proteins (recombinant CR domain, the ACP-CR didomain, and NanE) tested, only NanE hydrolyzed the polyether-SNAC. By contrast, recombinant DEBS TE from the erythromycin pathway, and the recombinant MonAX, a type II TE associated with the polyether monensin biosynthesis for which a homolog has not been detected in the nanchangmycin cluster, hydrolyzed a diketide-SNAC but not the polyether-SNAC. We could thus conclude that NanE is a dedicated thioesterase mediating the specific release of the polyether chain during nanchangmycin biosynthesis.

The first three domains of the insulin receptor differ structurally from the insulin-like growth factor 1 receptor in the regions governing ligand specificity

The insulin receptor (IR) and the type-1 insulin-like growth factor receptor (IGF1R) are homologous multidomain proteins that bind insulin and IGF with differing specificity. Here we report the crystal structure of the first three domains (L1-CR-L2) of human IR at 2.3 A resolution and compare it with the previously determined structure of the corresponding fragment of IGF1R. The most important differences seen between the two receptors are in the two regions governing ligand specificity. The first is at the corner of the ligand-binding surface of the L1 domain, where the side chain of F39 in IR forms part of the ligand binding surface involving the second (central) beta-sheet. This is very different to the location of its counterpart in IGF1R, S35, which is not involved in ligand binding. The second major difference is in the sixth module of the CR domain, where IR contains a larger loop that protrudes further into the ligand-binding pocket. This module, which governs IGF1-binding specificity, shows negligible sequence identity, significantly more alpha-helix, an additional disulfide bond, and opposite electrostatic potential compared to that of the IGF1R.

Protein Kinase D Intracellular Localization and Activity Control Kinase D-interacting Substrate of 220-kDa Traffic through a Postsynaptic Density-95/Discs Large/Zonula Occludens-1-binding Motif

Protein kinase D (PKD) controls protein traffic from the trans-Golgi network (TGN) to the plasma membrane of epithelial cells in an isoform-specific manner. However, whether the different PKD isoforms could be selectively regulating the traffic of their specific substrates remains unexplored. We identified the C terminus of the different PKDs that constitutes a postsynaptic density-95/discs large/zonula occludens-1 (PDZ)-binding motif in PKD1 and PKD2, but not in PKD3, to be responsible for the differential control of kinase D-interacting substrate of 220-kDa (Kidins220) surface localization, a neural membrane protein identified as the first substrate of PKD1. A kinase-inactive mutant of PKD3 is only able to alter the localization of Kidins220 at the plasma membrane when its C terminus has been substituted by the PDZ-binding motif of PKD1 or PKD2. This isoform-specific regulation of Kidins220 transport might not be due to differences among kinase activity or substrate selectivity of the PKD isoenzymes but more to the adaptors bound to their unique C terminus. Furthermore, by mutating the autophosphorylation site Ser(916), located at the critical position -2 of the PDZ-binding domain within PKD1, or by phorbol ester stimulation, we demonstrate that the phosphorylation of this residue is crucial for Kidins220-regulated transport. We also discovered that Ser(916) trans-phosphorylation takes place among PKD1 molecules. Finally, we demonstrate that PKD1 association to intracellular membranes is critical to control Kidins220 traffic. Our findings reveal the molecular mechanism by which PKD localization and activity control the traffic of Kidins220, most likely by modulating the recruitment of PDZ proteins in an isoform-specific and phosphorylation-dependent manner.

Connective-tissue growth factor (CTGF) modulates cell signalling by BMP and TGF-beta.

Connective-tissue growth factor (CTGF) is a secreted protein implicated in multiple cellular events including angiogenesis, skeletogenesis and wound healing. It is a member of the CCN family of secreted proteins, named after CTGF, cysteine-rich 61 (CYR61), and nephroblastoma overexpressed (NOV) proteins. The molecular mechanism by which CTGF or other CCN proteins regulate cell signalling is not known. CTGF contains a cysteine-rich domain (CR) similar to those found in chordin and other secreted proteins, which in some cases have been reported to function as bone morphogenetic protein (BMP) and TGF-beta binding domains. Here we show that CTGF directly binds BMP4 and TGF-beta 1 through its CR domain. CTGF can antagonize BMP4 activity by preventing its binding to BMP receptors and has the opposite effect, enhancement of receptor binding, on TGF-beta 1. These results show that CTGF inhibits BMP and activates TGF-beta signals by direct binding in the extracellular space.

BMP-binding modules in chordin: a model for signalling regulation in the extracellular space.

A number of genetic and molecular studies have implicated Chordin in the regulation of dorsoventral patterning during gastrulation. Chordin, a BMP antagonist of 120 kDa, contains four small (about 70 amino acids each) cysteine-rich domains (CRs) of unknown function. In this study, we show that the Chordin CRs define a novel protein module for the binding and regulation of BMPs. The biological activity of Chordin resides in the CRs, especially in CR1 and CR3, which have dorsalizing activity in Xenopus embryo assays and bind BMP4 with dissociation constants in the nanomolar range. The activity of individual CRs, however, is 5- to 10-fold lower than that of full-length Chordin. These results shed light on the molecular mechanism by which Chordin/BMP complexes are regulated by the metalloprotease Xolloid, which cleaves in the vicinity of CR1 and CR3 and would release CR/BMP complexes with lower anti-BMP activity than intact Chordin. CR domains are found in other extracellular proteins such as procollagens. Full-length Xenopus procollagen IIA mRNA has dorsalizing activity in embryo microinjection assays and the CR domain is required for this activity. Similarly, a C. elegans cDNA containing five CR domains induces secondary axes in injected Xenopus embryos. These results suggest that CR modules may function in a number of extracellular proteins to regulate growth factor signalling.

Sequence and expression of a rat cDNA for LECAM-1

A rat cDNA clone encoding an adhesion molecule, LECAM-1, has been isolated from the SD rat and the partial nucleotide sequence was determined. It encodes a peptide of 372 amino acids (aa), including a signal peptide of 38 aa. The protein has three tandem domains: a lectin domain, an EGF-like domain and two repeats of complement regulatory proteins (CR domain). The lectin binding domain has 93.2% and 81.4% and the EGF-like domain has 85.3% and 76.5% aa identity with those of mouse and human, respectively. In the CR repeat domain, the amino acid identity was 72.6% between human and rat and 71.8% between mouse and rat. Northern blot analysis detects the main transcript of about 3 kb in peripheral blood mononuclear cells (PBMC), spleen and thymus. The expression was down-regulated by mitogen stimulation of PBMC and spleen T cells. The protein encoded by this cDNA interacted with PPME when it was expressed on gp90 MEL-14 negative mouse EL-4 cells.

A generalised validity condition for local thermodynamic equilibrium in a laser-produced plasma

A generalised validity condition for local thermodynamic equilibrium (LTE) relevant to a laser-produced plasma is proposed and this new validity criterion is used to calculate the LTE domain for an aluminium-plasma. The applicability of the various equilibrium models, namely LTE, collisional-radiative model (CR) and coronal equilibrium (CE) is then checked for such a plasma. It is found that for a laser pulse of 1 J, 300 ps pulse width, and 3*1013 W cm-2 peak power, irradiating an aluminium target, more than 87% of the X-ray radiation is emitted for the CR domain of the plasma.