Bacteria Lectin Recognition Towards Fucose Binding Motifs Highlights the Impact of Presenting Mucin Core Glycopeptides

Abstract Dysregulation of transcriptional control is a common phenomenon associated with oncogenesis. Inhibitors of DNA binding (ID) proteins are critical actors in lymphopoiesis, acting as regulators of transcription through a helix-loop-helix (HLH) domain which enables heterodimerization with basic HLH (bHLH) proteins inhibiting their binding to DNA. ID proteins have been implicated in malignant transformation, but their role in multiple myeloma (MM) is unknown. Here, we evaluated the role of ID proteins in biology and transcriptional dysregulation in MM. We first evaluated the expression of the four ID proteins in normal and malignant plasma cells using RNA sequencing data from a cohort of 360 newly diagnosed MM patients and 16 normal plasma cells. We observed significant downregulation of ID2 in primary patient MM cells in comparison to normal plasma cells (p 0.0013). To study ID2 function in MM cells, we next overexpressed ID2 in 2 MM cell lines (MM1S and NCIH929) and observed a significant decrease in proliferation rate, together with G0/G1 phase cell cycle arrest. We performed RNA-sequencing to evaluate the transcriptomic changes following ID2 overexpression. Gene set enrichment analysis (GSEA) revealed significant downregulation of genes involved in E2F pathway and significant changes in pathways related to immune response, regulation of cell death and cell proliferation. In addition, analysis of upstream cis-regulatory motifs of genes significantly dysregulated in both cell lines (>1.5 fold change) showed a highly significant enrichment for bHLH class I transcription factors (E proteins) binding motifs. Conversely, stable ID2 knockdown in 4 MM cell lines (MM1S, NCIH929, RPMI8226 and KMS11) expressing intermediate levels of ID2, showed an increased proliferation rate, assessed by cell counting, H3-thymidine incorporation and ATP production. RNA-sequencing after ID2 knockdown in MM1S and NCIH929 cells showed 600 common genes upregulated in both cell lines (>1.5 fold change). GSEA revealed upregulation of pathways involved in inflammatory response and epithelial-to-mesenchymal transition, while upstream cis regulatory motifs analysis showed a highly significant enrichment for binding motifs of bHLH class I transcription factors E proteins, in particular Tcf3 (p <0.0001). Next, we sought to investigate the mechanisms involved in ID2 downregulation in MM. Since the role of the microenvironment is critical in myelomagenesis, we evaluated the impact of BM microenvironment on ID2 expression in a co-culture system. Using bone marrow stromal cells (BMSC) derived from MM patients and stromal cell line (HS5) in co-culture with various MM cell lines, we observed that both cell-cell interactions and soluble factors secreted by BMSC or HS5 were able to significantly downregulate ID2 expression at the RNA and protein level. Furthermore, ID2 overexpression in MM cell lines (MM1S and NCIH929) abrogated the impact of BMSC on MM cell proliferation. Next, we evaluated ID2 promoter methylation profile and binding motifs using Sequenom mass array and the assay for transposase-accessible chromatin sequencing (ATAC-seq), respectively. While we didn't observe any increase in methylation of CpG islands located in ID2 promoter in co-culture, explaining ID2 downregulation, we identified several binding motifs corresponding to known driver transcription factors in MM. Especially, we identified SP1 binding motif and we confirmed SP1 binding to ID2 promoter by ChIP-sequencing in MM1S, NCIH929 and U266. These data demonstrate that in MM, ID2 acts as a tumor suppressor by promoting major transcriptomic changes and cell cycle arrest. Bone marrow stromal cells further induce significant downregulation of ID2 in myeloma cells suggesting that ID2/bHLH axis and other ID2 related pathways represent a potential new therapeutic target in myeloma. Disclosures Anderson: Gilead: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Consultancy; Celgene: Consultancy; C4 Therapeutics: Equity Ownership, Other: Scientific founder; OncoPep: Equity Ownership, Other: Scientific founder; Millennium Takeda: Consultancy. Munshi:OncoPep: Other: Board of director.

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Frontispiece: The Impact of Heteromultivalency in Lectin Recognition and Glycosidase Inhibition: An Integrated Mechanistic Study

Chemistry - A European Journal ◽

10.1002/chem.201782663 ◽

2017 ◽

Vol 23 (26) ◽

Author(s):

M. Isabel García-Moreno ◽

Fernando Ortega-Caballero ◽

Rocío Rísquez-Cuadro ◽

Carmen Ortiz Mellet ◽

José M. García Fernández

Keyword(s):

Mechanistic Study ◽

The Impact ◽

Lectin Recognition ◽

Glycosidase Inhibition

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The Impact of Heteromultivalency in Lectin Recognition and Glycosidase Inhibition: An Integrated Mechanistic Study

Chemistry - A European Journal ◽

10.1002/chem.201700470 ◽

2017 ◽

Vol 23 (26) ◽

pp. 6295-6304 ◽

Cited By ~ 30

Author(s):

M. Isabel García-Moreno ◽

Fernando Ortega-Caballero ◽

Rocío Rísquez-Cuadro ◽

Carmen Ortiz Mellet ◽

José M. García Fernández

Keyword(s):

Mechanistic Study ◽

The Impact ◽

Lectin Recognition ◽

Glycosidase Inhibition

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Design–functionality relationships for adhesion/growth-regulatory galectins

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1813515116 ◽

2019 ◽

Vol 116 (8) ◽

pp. 2837-2842 ◽

Cited By ~ 24

Author(s):

Anna-Kristin Ludwig ◽

Malwina Michalak ◽

Qi Xiao ◽

Ulrich Gilles ◽

Francisco J. Medrano ◽

...

Keyword(s):

Therapeutic Potential ◽

Growth Inhibitor ◽

Modular Architecture ◽

The Family ◽

Specific Contact ◽

Galectin 3 ◽

Marked Selectivity ◽

Functional Antagonism ◽

The Impact ◽

Lectin Recognition

Glycan-lectin recognition is assumed to elicit its broad range of (patho)physiological functions via a combination of specific contact formation with generation of complexes of distinct signal-triggering topology on biomembranes. Faced with the challenge to understand why evolution has led to three particular modes of modular architecture for adhesion/growth-regulatory galectins in vertebrates, here we introduce protein engineering to enable design switches. The impact of changes is measured in assays on cell growth and on bridging fully synthetic nanovesicles (glycodendrimersomes) with a chemically programmable surface. Using the example of homodimeric galectin-1 and monomeric galectin-3, the mutual design conversion caused qualitative differences, i.e., from bridging effector to antagonist/from antagonist to growth inhibitor and vice versa. In addition to attaining proof-of-principle evidence for the hypothesis that chimera-type galectin-3 design makes functional antagonism possible, we underscore the value of versatile surface programming with a derivative of the pan-galectin ligand lactose. Aggregation assays with N,N′-diacetyllactosamine establishing a parasite-like surface signature revealed marked selectivity among the family of galectins and bridging potency of homodimers. These findings provide fundamental insights into design-functionality relationships of galectins. Moreover, our strategy generates the tools to identify biofunctional lattice formation on biomembranes and galectin-reagents with therapeutic potential.

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Before It Gets Started: Regulating Translation at the 5′ UTR

Comparative and Functional Genomics ◽

10.1155/2012/475731 ◽

2012 ◽

Vol 2012 ◽

pp. 1-8 ◽

Cited By ~ 110

Author(s):

Patricia R. Araujo ◽

Kihoon Yoon ◽

Daijin Ko ◽

Andrew D. Smith ◽

Mei Qiao ◽

...

Keyword(s):

Rna Binding ◽

Rna Binding Proteins ◽

Regulatory Elements ◽

Open Reading Frame ◽

Upstream Open Reading Frame ◽

Translation Regulation ◽

Binding Motifs ◽

Reading Frame ◽

Physiological Conditions ◽

The Impact

Translation regulation plays important roles in both normal physiological conditions and diseases states. This regulation requires cis-regulatory elements located mostly in 5′ and 3′ UTRs and trans-regulatory factors (e.g., RNA binding proteins (RBPs)) which recognize specific RNA features and interact with the translation machinery to modulate its activity. In this paper, we discuss important aspects of 5′ UTR-mediated regulation by providing an overview of the characteristics and the function of the main elements present in this region, like uORF (upstream open reading frame), secondary structures, and RBPs binding motifs and different mechanisms of translation regulation and the impact they have on gene expression and human health when deregulated.

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Missense Mutations Located in Structural p53 DNA-Binding Motifs Are Associated With Extremely Poor Survival in Chronic Lymphocytic Leukemia

Journal of Clinical Oncology ◽

10.1200/jco.2011.34.7872 ◽

2011 ◽

Vol 29 (19) ◽

pp. 2703-2708 ◽

Cited By ~ 62

Author(s):

Martin Trbusek ◽

Jana Smardova ◽

Jitka Malcikova ◽

Ludmila Sebejova ◽

Petr Dobes ◽

...

Keyword(s):

Chronic Lymphocytic Leukemia ◽

Dna Binding ◽

Response To Therapy ◽

Lymphocytic Leukemia ◽

Functional Assay ◽

Missense Mutations ◽

P53 Mutations ◽

Binding Motifs ◽

Dna Binding Motifs ◽

The Impact

Purpose There is a distinct connection between TP53 defects and poor prognosis in chronic lymphocytic leukemia (CLL). It remains unclear whether patients harboring TP53 mutations represent a homogenous prognostic group. Patients and Methods We evaluated the survival of patients with CLL and p53 defects identified at our institution by p53 yeast functional assay and complementary interphase fluorescence in situ hybridization analysis detecting del(17p) from 2003 to 2010. Results A defect of the TP53 gene was identified in 100 of 550 patients. p53 mutations were strongly associated with the deletion of 17p and the unmutated IgVH locus (both P < .001). Survival assessed from the time of abnormality detection was significantly reduced in patients with both missense (P < .001) and nonmissense p53 mutations (P = .004). In addition, patients harboring missense mutation located in p53 DNA-binding motifs (DBMs), structurally well-defined parts of the DNA-binding domain, manifested a clearly shorter median survival (12 months) compared with patients having missense mutations outside DBMs (41 months; P = .002) or nonmissense alterations (36 months; P = .005). The difference in survival was similar in the analysis limited to patients harboring mutation accompanied by del(17p) and was also confirmed in a subgroup harboring TP53 defect at diagnosis. The patients with p53 DBMs mutation (at diagnosis) also manifested a short median time to first therapy (TTFT; 1 month). Conclusion The substantially worse survival and the short TTFT suggest a strong mutated p53 gain-of-function phenotype in patients with CLL with DBMs mutations. The impact of p53 DBMs mutations on prognosis and response to therapy should be analyzed in investigative clinical trials.

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Homology-mediated inter-chromosomal interactions in hexaploid wheat lead to specific subgenome territories following polyploidization and introgression

Genome Biology ◽

10.1186/s13059-020-02225-7 ◽

2021 ◽

Vol 22 (1) ◽

Author(s):

Jizeng Jia ◽

Yilin Xie ◽

Jingfei Cheng ◽

Chuizheng Kong ◽

Meiyue Wang ◽

...

Keyword(s):

Genome Evolution ◽

Hexaploid Wheat ◽

Wheat Variety ◽

Three Dimensional ◽

Higher Order ◽

Plant Genome ◽

Translocation Chromosome ◽

Binding Motifs ◽

Allohexaploid Wheat ◽

The Impact

Abstract Background Polyploidization and introgression are major events driving plant genome evolution and influencing crop breeding. However, the mechanisms underlying the higher-order chromatin organization of subgenomes and alien chromosomes are largely unknown. Results We probe the three-dimensional chromatin architecture of Aikang 58 (AK58), a widely cultivated allohexaploid wheat variety in China carrying the 1RS/1BL translocation chromosome. The regions involved in inter-chromosomal interactions, both within and between subgenomes, have highly similar sequences. Subgenome-specific territories tend to be connected by subgenome-dominant homologous transposable elements (TEs). The alien 1RS chromosomal arm, which was introgressed from rye and differs from its wheat counterpart, has relatively few inter-chromosome interactions with wheat chromosomes. An analysis of local chromatin structures reveals topologically associating domain (TAD)-like regions covering 52% of the AK58 genome, the boundaries of which are enriched with active genes, zinc-finger factor-binding motifs, CHH methylation, and 24-nt small RNAs. The chromatin loops are mostly localized around TAD boundaries, and the number of gene loops is positively associated with gene activity. Conclusions The present study reveals the impact of the genetic sequence context on the higher-order chromatin structure and subgenome stability in hexaploid wheat. Specifically, we characterized the sequence homology-mediated inter-chromosome interactions and the non-canonical role of subgenome-biased TEs. Our findings may have profound implications for future investigations of the interplay between genetic sequences and higher-order structures and their consequences on polyploid genome evolution and introgression-based breeding of crop plants.

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Simulations of Higher-Order Protein Organizations Using a Fuzzy Framework

Complexity ◽

10.1155/2018/6360846 ◽

2018 ◽

Vol 2018 ◽

pp. 1-10 ◽

Cited By ~ 3

Author(s):

B. Tüű-Szabó ◽

L. T. Kóczy ◽

M. Fuxreiter

Keyword(s):

Driving Forces ◽

Critical Role ◽

Critical Factor ◽

Higher Order ◽

Mathematical Framework ◽

Protein Assemblies ◽

Binding Motifs ◽

Spatiotemporal Regulation ◽

The Impact

Spatiotemporal regulation of the biochemical information is often linked to supramolecular organizations proteins and nucleic acids, the driving forces of which have yet to be elucidated. Although the critical role of multivalency in phase transition has been recognized, the organization principles of higher-order structures need to be understood. Here, we present a fuzzy mathematical framework to handle the heterogeneity of interactions patterns and the resultant multiplicity of conformational states in protein assemblies. In this model, redundant binding motifs can establish simultaneous and partial interactions with multiple targets. We demonstrate that these multivalent, weak contacts facilitate polymer formation, while recapitulating the observed valency-dependence. In addition, the impact of linker dynamics and motif binding affinity, as well as the interplay between the two effects was studied. Our results support that fuzziness is a critical factor in driving higher-order protein organizations, and this could be used as a general framework to simulate different kinds of supramolecular assemblies.

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Large Scale Screening for DNA Damage-Induced Transcription Factors as Potential Targets for Treatment of CLL with p53 Apoptotic Defect.

Blood ◽

10.1182/blood.v114.22.3439.3439 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3439-3439

Author(s):

Victoria Weston ◽

Paul Moss ◽

A. Malcolm R Taylor ◽

Tatjana Stankovic

Keyword(s):

Dna Damage ◽

Transcription Factors ◽

Dna Binding ◽

Aryl Hydrocarbon Receptor ◽

P53 Pathway ◽

Binding Motifs ◽

Aryl Hydrocarbon ◽

Dna Damaging Agents ◽

Dna Binding Motifs ◽

The Impact

Abstract Abstract 3439 Poster Board III-327 Chronic lymphocytic leukaemia (CLL) is a malignancy with a variable clinical course in which a proportion of patients exhibits rapid clinical progression despite treatment. One of the major causes of treatment resistance is alterations in the ATM/p53 pathway imposed by mutations in either the ATM or TP53 genes. Consequently, there is an urgent need to devise novel therapeutic approaches that will be able to counteract the p53 apoptotic defect in these tumours. We have previously shown that DNA damage induces a complex ATM-dependent network of pro-survival and pro-apoptotic transcriptional responses (both p53-dependent and -independent) and that the balance between these responses determines CLL cellular death. Therefore, it is plausible to expect that manipulation of ATM-dependent transcription to either reduce pro-survival or increase pro-apoptotic signals can sensitise ATM and TP53 mutant CLL tumours to DNA damaging agents. Individual transcription factors (TFs) that govern ATM-dependent transcription are largely unknown. In this study we aimed to identify those factors by employing a DNA/Protein Transcription Factor ComboArray (Panomics/Affymetrix) which includes 345 DNA binding motifs for a range of transcription factors, DNA binding proteins and response elements. We compared the ability of nuclear cell extracts from 3 combined ATM wildtype primary CLL samples and 3 combined ATM mutant primary CLL samples to bind to biotin-labelled DNA binding motifs prior to irradiation (IR)-induced DNA damage, 2h and 6h post-IR. Following hybridisation of nuclear protein-bound biotin-labelled probes to the array and HRP visualisation, we identified 49 binding motifs (several of which were detected more than once through alternative sequences) which, in response to DNA damage, exhibited reduced binding in ATM mutant compared to the ATM wildtype CLL nuclear extracts. The most prominent differentially bound DNA binding motifs included those for GATA1 and 2, Transcriptional enhancer factor 1 (TEF1), c-Rel, Aryl hydrocarbon receptor/aryl hydrocarbon receptor nuclear translocator binding element (AhR/Arnt), forkhead box I1 (HFH-3), Slow/Cardiac Troponin C (cTnC/CEF-2), E2A immunoglobulin enhancer binding factors (E12/E47), Pax-4, Wilms tumour 1 (WT1), antioxidant recognition element (ARE) and interferon-a stimulated response element (ISRE). We validated differential binding of individual TFs by electro-mobility shift analysis (EMSA) and selected six that were positively corroborated in an independent cohort of primary ATM mutant and ATM wildtype CLL tumour cells. We subsequently investigated the impact of altering the activity of the identified ATM-dependent TFs on the sensitivity of ATM mutant CLL tumours to DNA damage. Among the selected TFs, as a proof of principle, ARE demonstrated both ATM-dependent binding by EMSA as well as the capacity to modulate the DNA damage response in CLL cells: pharmacological activation of this TF by Dimethyl fumarate (DMF) sensitised ATM mutant cells to IR-induced DNA damage. In summary, we have identified a number of ATM-regulated transcription factors that could be directly or indirectly targeted to increase the sensitivity of CLL cells with a defective ATM/p53 pathway to DNA damaging agents. We also suggest that the DNA damage-dependent TF screen represents a feasible approach to identify novel molecular targets that may sensitise other subtypes of treatment-resistant CLL tumours. Disclosures No relevant conflicts of interest to declare.

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SARNAclust: Semi-automatic detection of RNA protein binding motifs from immunoprecipitation data

10.1101/127878 ◽

2017 ◽

Author(s):

Ivan Dotu ◽

Scott Adamson ◽

Benjamin Coleman ◽

Cyril Fournier ◽

Emma Ricart-Altimiras ◽

...

Keyword(s):

Protein Binding ◽

Protein Interactions ◽

Difficult Problem ◽

Sequence Motifs ◽

Rna Motifs ◽

Binding Motifs ◽

Combined Structure ◽

Aberrant Rna ◽

The Impact ◽

Automatic Algorithm

AbstractRNA-protein binding is critical to gene regulation, controlling fundamental processes including splicing, translation, localization and stability, and aberrant RNA-protein interactions are known to play a role in a wide variety of diseases. However, molecular understanding of RNA-protein interactions remains limited, and in particular identification of the RNA motifs that bind proteins has long been a difficult problem. To address this challenge, we have developed a novel semi-automatic algorithm, SARNAclust, to computationally identify combined structure/sequence motifs from immunoprecipitation data. SARNAclust is, to our knowledge, the first unsupervised method that can identify RNA motifs at full structural resolution while also being able to simultaneously deconvolve multiple motifs. SARNAclust makes use of a graph kernel to evaluate similarity between sequence/structure objects, and provides the ability to isolate the impact of specific features through the bulge graph formalism. SARNAclust includes a key method for predicting RNA secondary structure at CLIP peaks, RNApeakFold, which we have verified to be effective on synthetic motif data. We applied SARNAclust to 30 ENCODE eCLIP datasets, identifying known motifs and novel predictions. Notably, we predicted a new motif for the protein ILF3 similar to that for the splicing factor hnRNPC, providing evidence for interaction between these two proteins. To validate our predictions, we performed a directed RNA bind-n-seq assay for two proteins: ILF3 and SLBP, in each case revealing the effectiveness of SARNAclust in predicting RNA sequence and structure elements important to protein binding. Availability: https://github.com/idotu/SARNAclust

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