Promiscuous Interactions Research Articles

Expansionof the evolutionarily conserved network of J-domain proteins in the Arabidopsis mitochondrial import complex.

We report that discriminate interaction between the expanded mitochondrial chaperone network and variability in their expression might determine their functional specificities and impart robustness to mitochondrial import processes in plants. Mitochondrial Hsp70 (mtHsp70), the central component of the pre-sequence associated motor (PAM) complex, is crucial for the import of proteins to the mitochondrial matrix. Activity of mtHsp70 is regulated by a heterodimeric complex of two J-domain proteins (JDPs), Pam18 and Pam16. Compared to other eukaryotes, plants harbor multiple copies of these JDPs, which posit that plants have an increasingly complex mtHsp70: JDPnetwork in their mitochondrial matrix. Here, we show that although highly similar in sequence, some of the plant JDPs are functionally different. Protein: protein interaction studies including yeast two-hybrid and Bimolecular Fluorescence Complementation revealed that while all the AtPam18s interacted with AtPam16s, the strengths of these promiscuous interactions are variable. Further, down-regulation ofAtPAM16L affected seed germination, even in the presence of its seemingly identical paralog, AtPAM16. Knockdown ofAtPAM16L caused reduction in mitochondrial number and deregulation of several mitochondrial genes, suggesting towards a specific role ofAtPam16L in maintaining mitochondrial homeostasis, especially under stress conditions. Our findings suggest that variations in the spatio-temporal expression, accompanied by discriminate interactions between the JDPs, might be defining the functional specificity of the mtHsp70 co-chaperone machinery and providing resilience to mitochondrial import processes in plants, especially under stress conditions.

TssA\u2013TssM\u2013TagA interaction modulates type VI secretion system sheath-tube assembly in Vibrio cholerae

The type VI protein secretion system (T6SS) is a powerful needle-like machinery found in Gram-negative bacteria that can penetrate the cytosol of receiving cells in milliseconds by physical force. Anchored by its membrane-spanning complex (MC) and a baseplate (BP), the T6SS sheath-tube is assembled in a stepwise process primed by TssA and terminated by TagA. However, the molecular details of its assembly remain elusive. Here, we systematically examined the initiation and termination of contractile and non-contractile T6SS sheaths in MC-BP, tssA and tagA mutants by fluorescence microscopy. We observe long pole-to-pole sheath-tube structures in the non-contractile MC-BP defective mutants but not in the Hcp tube or VgrG spike mutants. Combining overexpression and genetic mutation data, we demonstrate complex effects of TssM, TssA and TagA interactions on T6SS sheath-tube dynamics. We also report promiscuous interactions of TagA with multiple T6SS components, similar to TssA. Our results demonstrate that priming of the T6SS sheath-tube assembly is not dependent on TssA, nor is the assembly termination dependent on the distal end TssA–TagA interaction, and highlight the tripartite control of TssA–TssM–TagA on sheath-tube initiation and termination.

Configuration-Controlled Crystal and/or Gel Formation of Protected d-Glucosamines Supported by Promiscuous Interaction Surfaces and a Conformationally Heterogeneous Solution State.

The configuration-dependent self-association mode of the two anomers of O-Ac,N-Fmoc-d-glucosamine, a foldamer building block, leading to gel and/or single crystal formation is described. The β-anomer of the sugar amino acid (2) forms a gel from various solvents (confirmed by SEM, rheology measurements, NMR, and ECD spectroscopy), whereas the α-anomer (1) does not form a gel with any solvent tested. Transition from the solution state to a gel is coupled to a concurrent shift of the Fmoc-groups: from a freely rotating (almost symmetrical) to a specific, asymmetric orientation. Whereas the crystal structure of the α-anomer is built as an evenly packed 3D system, the β-anomer forms a looser superstructure of well-packed 2D layers. Modeling indicates that in the lowest energy, but scarcely sampled conformer of the β-anomer, the Fmoc-group bends above the sugar moiety, stabilized by intramolecular CH↔π interactions between the aromatic rings. It is concluded that possessing an extended and promiscuous interaction surface and a conformationally heterogeneous solution state are among the basic requirements of gel formation for a candidate molecule.

Weak Microbial Metabolites: a Treasure Trove for Using Biomimicry to Discover and Optimize Drugs.

For decades, traditional drug discovery has used natural product and synthetic chemistry approaches to generate libraries of compounds, with some ending as promising drug candidates. A complementary approach has been to adopt the concept of biomimicry of natural products and metabolites so as to improve multiple drug-like features of the parent molecule. In this effort, promiscuous and weak interactions between ligands and receptors are often ignored in a drug discovery process. In this Emerging Concepts article, we highlight microbial metabolite mimicry, whereby parent metabolites have weak interactions with their receptors that then have led to discrete examples of more potent and effective drug-like molecules. We show specific examples of parent-metabolite mimics with potent effects in vitro and in vivo. Furthermore, we show examples of emerging microbial ligand-receptor interactions and provide a context in which these ligands could be improved as potential drugs. A balanced conceptual advance is provided in which we also acknowledge potential pitfalls-hyperstimulation of finely balanced receptor-ligand interactions could also be detrimental. However, with balance, we provide examples of where this emerging concept needs to be tested. SIGNIFICANCE STATEMENT: Microbial metabolite mimicry is a novel way to expand on the chemical repertoire of future drugs. The emerging concept is now explained using specific examples of the discovery of therapeutic leads from microbial metabolites.

Proteinaceous Transformers: Structural and Functional Variability of Human sHsps.

The proteostasis network allows organisms to support and regulate the life cycle of proteins. Especially regarding stress, molecular chaperones represent the main players within this network. Small heat shock proteins (sHsps) are a diverse family of ATP-independent molecular chaperones acting as the first line of defense in many stress situations. Thereby, the promiscuous interaction of sHsps with substrate proteins results in complexes from which the substrates can be refolded by ATP-dependent chaperones. Particularly in vertebrates, sHsps are linked to a broad variety of diseases and are needed to maintain the refractive index of the eye lens. A striking key characteristic of sHsps is their existence in ensembles of oligomers with varying numbers of subunits. The respective dynamics of these molecules allow the exchange of subunits and the formation of hetero-oligomers. Additionally, these dynamics are closely linked to the chaperone activity of sHsps. In current models a shift in the equilibrium of the sHsp ensemble allows regulation of the chaperone activity, whereby smaller oligomers are commonly the more active species. Different triggers reversibly change the oligomer equilibrium and regulate the activity of sHsps. However, a finite availability of high-resolution structures of sHsps still limits a detailed mechanistic understanding of their dynamics and the correlating recognition of substrate proteins. Here we summarize recent advances in understanding the structural and functional relationships of human sHsps with a focus on the eye-lens αA- and αB-crystallins.

Phylogeny, evolution, and potential ecological relationship of cytochrome CYP52 enzymes in Saccharomycetales yeasts

Cytochrome P450s from the CYP52 family participate in the assimilation of alkanes and fatty acids in fungi. In this work, the evolutionary history of a set of orthologous and paralogous CYP52 proteins from Saccharomycetales yeasts was inferred. Further, the phenotypic assimilation profiles were related with the distribution of cytochrome CYP52 members among species. The maximum likelihood phylogeny of CYP52 inferred proteins reveled a frequent ancient and modern duplication and loss events that generated orthologous and paralogous groups. Phylogeny and assimilation profiles of alkanes and fatty acids showed a family expansion in yeast isolated from hydrophobic-rich environments. Docking analysis of deduced ancient CYP52 proteins suggests that the most ancient function was the oxidation of C4-C11 alkanes, while the oxidation of >10 carbon alkanes and fatty acids is a derived character. The ancient CYP52 paralogs displayed partial specialization and promiscuous interaction with hydrophobic substrates. Additionally, functional optimization was not evident. Changes in the interaction of ancient CYP52 with different alkanes and fatty acids could be associated with modifications in spatial orientations of the amino acid residues that comprise the active site. The extended family of CYP52 proteins is likely evolving toward functional specialization, and certain redundancy for substrates is being maintained.

Intrinsically Disordered Regions Direct Transcription Factor In Vivo Binding Specificity

Transcription factors (TFs) that bind common DNA motifs invitro occupy distinct sets of promoters invivo, raising the question of how binding specificity is achieved. TFs are enriched with intrinsically disordered regions (IDRs). Such regions commonly form promiscuous interactions, yet their unique properties might also benefit specific binding-site selection. We examine this using Msn2 and Yap1, TFs of distinct families that contain long IDRs outside their DNA-binding domains. We find that these IDRs are both necessary and sufficient for localizing to the majority of target promoters. This IDR-directed binding does not depend on any localized domain but results from a multitude of weak determinants distributed throughout the entire IDR sequence. Furthermore, IDR specificity is conserved between distant orthologs, suggesting direct interaction with multiple promoters. We propose that distribution of sensing determinants along extended IDRs accelerates binding-site detection by rapidly localizing TFs to broad DNA regions surrounding these sites.

Influence of the geometry of fluorescently labelled DNA constructs on fluorescence anisotropy assay

DNA-encoded chemical libraries (DECLs) are powerful tools for modern drug discovery. A DECL is a pooled mixture of small molecule compounds, each of which is tagged with a unique DNA sequence which functions as a barcode. After incubation with a drug target and washing to remove non-binders, the bound molecules are eluted and submitted for DNA sequencing to determine which molecules are binding the target. While the DECL technology itself is ultra-high throughput, the following re-synthesis of identified compounds for orthogonal validation experiments remains the bottleneck. Using existing DNA-small molecule conjugates directly for affinity measurements, as opposed to complete compound resynthesis, could accelerate the discovery process. To this end, we have tested various geometries of fluorescently-labelled DNA constructs for fluorescence anisotropy (FA) experiments. Minimizing the distance between the fluorescent moiety and ligand can maximize the correlation between ligand-protein interaction and corresponding change in fluorophore rotational freedom, thus leading to larger, easier to interpret changes in FA values. However, close proximity can also cause artifacts due to potentially promiscuous interactions between fluorophore and protein. By balancing these two opposite effects, we have identified applicable fluorescently labelled DNA constructs displaying either a single ligand or pairs of fragments for affinity measurement using a FA assay.

The Role of the FMN-Domain of Human Cytochrome P450 Oxidoreductase in Its Promiscuous Interactions With Structurally Diverse Redox Partners.

NADPH cytochrome P450 oxidoreductase (CPR) is the obligatory electron supplier that sustains the activity of microsomal cytochrome P450 (CYP) enzymes. The variant nature of the isoform-specific proximal interface of microsomal CYPs indicates that CPR is capable of multiple degenerated interactions with CYPs for electron transfer, through different binding mechanisms, and which are still not well-understood. Recently, we showed that CPR dynamics allows formation of open conformations that can be sampled by its structurally diverse redox partners in a CYP-isoform dependent manner. To further investigate the role of the CPR FMN-domain in effective binding of CPR to its diverse acceptors and to clarify the underlying molecular mechanisms, five different CPR-FMN-domain random mutant libraries were created. These libraries were screened for mutants with increased activity when combined with specific CYP-isoforms. Seven CPR-FMN-domain mutants were identified, supporting a gain in activity for CYP1A2 (P117H, G144C, A229T), 2A6 (P117L/L125V, G175D, H183Y), or 3A4 (N151D). Effects were evaluated using extended enzyme kinetic analysis, cytochrome b5 competition, ionic strength effect on CYP activity, and structural analysis. Mutated residues were located either in or adjacent to several acidic amino acid stretches – formerly indicated to be involved in CPR:CYP interactions – or close to two tyrosine residues suggested to be involved in FMN binding. Several of the identified positions co-localize with mutations found in naturally occurring CPR variants that were previously shown to cause CYP-isoform-dependent effects. The mutations do not seem to significantly alter the geometry of the FMN-domain but are likely to cause very subtle alterations leading to improved interaction with a specific CYP. Overall, these data suggest that CYPs interact with CPR using an isoform specific combination of several binding motifs of the FMN-domain.

Broad Specificity of Amino Acid Chemoreceptor CtaA of Pseudomonas fluorescens Is Afforded by Plasticity of Its Amphipathic Ligand-Binding Pocket.

Motile bacteria follow gradients of nutrients or other environmental cues. Many bacterial chemoreceptors that sense exogenous amino acids contain a double Cache (dCache; calcium channels and chemotaxis receptors) ligand-binding domain (LBD). A growing number of studies suggest that broad-specificity dCache-type receptors that sense more than one amino acid are common. Here, we present an investigation into the mechanism by which the dCache LBD of the chemoreceptor CtaA from a plant growth-promoting rhizobacterium, Pseudomonas fluorescens, recognizes several chemically distinct amino acids. We established that amino acids that signal by directly binding to the CtaA LBD include ones with aliphatic (l-alanine, l-proline, l-leucine, l-isoleucine, l-valine), small polar (l-serine), and large charged (l-arginine) side chains. We determined the structure of CtaA LBD in complex with different amino acids, revealing that its ability to recognize a range of structurally and chemically distinct amino acids is afforded by its easily accessible plastic pocket, which can expand or contract according to the size of the ligand side chain. The amphipathic character of the pocket enables promiscuous interactions with both polar and nonpolar amino acids. The results not only clarify the means by which various amino acids are recognized by CtaA but also reveal that a conserved mobile lid over the ligand-binding pocket adopts the same conformation in all complexes, consistent with this being an important and invariant part of the signaling mechanism.

Tripartite phase separation of two signal effectors with vesicles priming B cell responsiveness

Antibody-mediated immune responses rely on antigen recognition by the B cell antigen receptor (BCR) and the proper engagement of its intracellular signal effector proteins. Src homology (SH) 2 domain-containing leukocyte protein of 65 kDa (SLP65) is the key scaffold protein mediating BCR signaling. In resting B cells, SLP65 colocalizes with Cbl-interacting protein of 85 kDa (CIN85) in cytoplasmic granules whose formation is not fully understood. Here we show that effective B cell activation requires tripartite phase separation of SLP65, CIN85, and lipid vesicles into droplets via vesicle binding of SLP65 and promiscuous interactions between nine SH3 domains of the trimeric CIN85 and the proline-rich motifs (PRMs) of SLP65. Vesicles are clustered and the dynamical structure of SLP65 persists in the droplet phase in vitro. Our results demonstrate that phase separation driven by concerted transient interactions between scaffold proteins and vesicles is a cellular mechanism to concentrate and organize signal transducers.

Burden of Functional Features and Genetic Variations in Human Intrinsically Disordered Proteins

Several physicochemical and structural properties of intrinsically disordered protein (IDP) and disordered regions (IDR) are now well-established (e.g. high net-charge, low hydrophobicity). Functional characterization of IDPs and IDRs, however, are challenging due to their highly dynamic character, promiscuous interaction, and coupled folding and binding. Current open-source databases enable us to perform data-driven inference of disorder residues functional features, which may link to their role in disease etiology. In this study, we applied two-tailed Fisher's exact test to compute the burden of UniProt-based sequence annotations in disordered (N=24,097) versus ordered (N=80,069) residues of 210 human IDPs from DisProt7.0 database. We identified 6 (out of 25) features enriched for disordered residues: modified residues (p<1.00e-100), DNA-binding sites (p<1.00e-100), motifs (p<1.00e-100), cross-links (p=5.09e-08), region (p<1.00e-100), and repeat (p=2.41e-10). The test could recapitulate several established signature properties of ordered residues as well, such as transmembrane domain (p<1.00e-100) and disulfide bond (p<1.00e-100). Separate analyses on 58 haploinsufficient genes and 24 genes under selection against amino acid substitutions, we found two more disorder-enriched features, namely, propeptide (p=6.53e-05) and zinc finger (p=2.73e-06). We then investigated the genetic variants from the genome aggregation database (gnomAD), and observed an enrichment of frameshift (N=344,1.51-fold) and in-frame insertion/deletion (N=258, 1.86-fold) mutations compared to the silent mutations in IDPs. Notably, we observed a prevalence of rare singleton frameshift (69.2%) and in-frame (53.5%) mutations in IDRs with different disorder-enriched features. We performed association analysis to identify statistically significant functional features of disordered residues, compared to the ordered residues, and explored the link among disorder, function, and genomic variation. Leveraging high-fidelity predictions, we aim to apply our method at higher scale for genome-wide characterization of IDPs, which may reveal novel insights and help in designing experiments to identify contribution of IDPs in pathology.

Dynamics and Location of the Allosteric Midazolam Site in Cytochrome P4503A4 in Lipid Nanodiscs

Promiscuous and allosteric drug interactions with cytochrome P450 3A4 (CYP3A4) are ubiquitous but incompletely understood at the molecular level. A classic allosteric CYP3A4 drug interaction includes the benzodiazepine midazolam (MDZ). MDZ exhibits homotropic and heterotropic allostery when metabolized to 1'-hydroxy and 4-hydroxy metabolites in varying ratios. The combination of hydrogen-deuterium exchange mass spectrometry (HDX-MS) and Gaussian accelerated molecular dynamics (GaMD) simulations of CYP3A4 in lipid nanodiscs and in a lipid bilayer, respectively, reveals MDZ-dependent changes in dynamics in a membrane environment. The F-, G-, and intervening helices, as well as the loop preceding the β1-sheets, display the largest observed changes in HDX. The GaMD suggests a potential allosteric binding site for MDZ in the F'- and G'-regions, which undergo significant increases in HDX at near-saturating MDZ concentrations. The HDX-MS and GaMD results confirm that changes in dynamics are most significant near the developing consensus allosteric site, and these changes are distinct from those observed previously with the nonallosteric inhibitor ketoconazole. The results suggest that the allosteric MDZ remains mobile in its binding site at the Phe-cluster. The results further suggest that this binding site remains dynamic or changes the depth of insertion in the membrane.

Sorting nexin 27 (SNX27) variants associated with seizures, developmental delay, behavioral disturbance, and subcortical brain abnormalities.

Sorting nexin 27 (SNX27) influences the composition of the cellular membrane via regulation of selective endosomal recycling. Molecular analysis indicates that SNX27 regulates numerous cellular processes through promiscuous interactions with its receptor cargos. SNX27 deficient (Snx27 -/- ) mice exhibit reduced embryonic survival, marked postnatal growth restriction and lethality. Haploinsufficient mice (Snx27 +/- ) show a less severe phenotype, with deficits in learning, memory, synaptic transmission and neuronal plasticity. One family previously reported with a homozygous SNX27 frameshift variant (c.515_516del;p.His172Argfs*6), exhibited infantile intractable myoclonic epilepsy, axial hypotonia, startle-like movements, cardiac septal defects, global developmental delay, failure to thrive, recurrent chest infections, persistent hypoxemia and early death secondary to respiratory failure. Here, we report two additional patients with compound heterozygous SNX27 variants, that are predicted to be damaging: (a) c.510C>G;p.Tyr170* and c.1295G>A;p.Cys432Tyr, and (b) c.782dupT;p.Leu262Profs*6 and c.989G>A;p.Arg330His. They exhibit global developmental delay, behavioral disturbance, epilepsy, some dysmorphic features and subcortical white matter abnormalities. In addition, possible connective tissue involvement was noted. Epilepsy, developmental delays and subcortical white matter abnormalities appear to be core features of SNX27-related disorders. We correlate the observed phenotype with available in vitro, in vivo and proteomic data and suggest additional possible molecular mediators of SNX27-related pathology.

Using evasins to target the chemokine network in inflammation.

Inflammation, is driven by a network comprising cytokines, chemokines, their target receptors and leukocytes, and is a major pathologic mechanism that adversely affects organ function in diverse human diseases. Despite being supported by substantial target validation, no successful anti-chemokine therapeutic to treat inflammatory disease has yet been developed. This is in part because of the robustness of the chemokine network, which emerges from a large total chemokine load in disease, promiscuous expression of receptors on leukocytes, promiscuous and synergistic interactions between chemokines and receptors, and feedforward loops created by secretion of chemokines by leukocytes themselves. Many parasites, including viruses, helminths and ticks, evade the chemokine network by producing proteins that bind promiscuously to chemokines or their receptors. Evasins - three small glycoproteins identified in the saliva of the brown dog tick - bind multiple chemokines, and are active in several animal models of inflammatory disease. Over 50 evasin homologs have recently been identified from diverse tick species. Characterization of the chemokine binding patterns of evasins show that several have anti-chemokine activities that extend substantially beyond those previously described. These studies indicate that evasins function at the site of the tick bite by reducing total chemokine load. This not only reduces chemokine signaling to receptors, but also interrupts feedforward loops, thus disabling the chemokine network. Taking the lead from nature, a goal for the development of new anti-chemokine therapeutics would be to reduce the total chemokine load in disease. This could be achieved by administering appropriate evasin combinations or by smaller peptides that mimic evasin action.

RNA is a critical element for the sizing and the composition of phase-separated RNA\u2013protein condensates

Liquid–liquid phase separation is thought to be a key organizing principle in eukaryotic cells to generate highly concentrated dynamic assemblies, such as the RNP granules. Numerous in vitro approaches have validated this model, yet a missing aspect is to take into consideration the complex molecular mixture and promiscuous interactions found in vivo. Here we report the versatile scaffold ArtiG to generate concentration-dependent RNA–protein condensates within living cells, as a bottom-up approach to study the impact of co-segregated endogenous components on phase separation. We demonstrate that intracellular RNA seeds the nucleation of the condensates, as it provides molecular cues to locally coordinate the formation of endogenous high-order RNP assemblies. Interestingly, the co-segregation of intracellular components ultimately impacts the size of the phase-separated condensates. Thus, RNA arises as an architectural element that can influence the composition and the morphological outcome of the condensate phases in an intracellular context.

Abstract LB-109: A novel therapeutic modality of inhibiting the glyco-immune checkpoint axis to treat cancer

Abstract Cancer therapy has been revolutionized by the recent developments of immune-checkpoint inhibitors (ICI) to harness the power of the immune system in fighting cancer. However, most patients fail to have durable responses or become resistant to ICI, highlighting the need to identify new mechanisms of immune evasion in cancer and develop novel therapeutic modalities. Recently, the glyco-immune checkpoint axis (sialoglycan/Siglec pathway) has emerged as a new mechanism of immune regulation involving both innate and adaptive immunity and an important mechanism of cancer immune escape. Upon ligation of sialoglycan to ITIM-containing Siglecs on immune cells, this pathway suppresses multiple facets of anti-cancer immunity, including cancer antigen release, cancer antigen presentation, and priming and activation of anti-cancer T cells. However, a therapeutic intervention of this axis remains a great challenge due to the overlapping and promiscuous receptor-ligand interactions between 15 Siglecs and dense array of various sialoglycans in humans. To overcome this hurdle and block the glyco-immune checkpoint axis, we describe here a new therapeutic modality named EAGLE (Enzyme-Antibody Glyco-Ligand Editing), which is antibody-like, multi-functional, and comprised of a tumor-associated antigen-binding moiety and a sialidase moiety, allowing selectively removing terminal sialic acids, the critical binding carbohydrate of Siglecs, from sialoglycans on tumor cells. We demonstrated that EAGLE decreased sialic acid levels of tumor cells and enhanced anti-tumor immune responses using multiple human system models mimicking immunosuppressive tumor microenvironment and immunocompetent syngeneic mouse tumor models. EAGLE treatment released cancer cell-mediated immunosuppression, restored dendritic cell functions, enhanced CD8+ T-cell proliferation/activation, and induced proinflammatory cytokines IFNγ, IL-17A, IL-2, IL-6, and TNFα in human coculture assays of cancer cells with dendritic cells or PBMC in the presence or absence of primary endothelial cells. Systematic administration of EAGLE increased tumor-infiltrating immune cells and led to significant anti-tumor activities with complete regressions as monotherapy in syngeneic mouse tumor models. Re-challenge experiments in cured mice from the EAGLE treatment resulted in a complete rejection of tumor cells, demonstrating that EAGLE induced anti-tumor immunological memory. We further revealed that the mechanism of action of EAGLE involved both innate and adaptive immunity because depleting macrophages or CD8+ T-cells decreased or abolished its efficacy. Moreover, EAGLE in combination with anti-PD1 mAb treatment achieved ~100% cures in syngeneic EMT6-Her2 models. In summary, EAGLE is a novel and promising immunomodulatory therapeutic modality inhibiting the glyco-immune checkpoints and has the potential to overcome resistance to current immunotherapies. Citation Format: Lizhi Cao, Adam Petrone, Wayne Gatlin, Jenny Che, Abhishek Das, Robert LeBlanc, Zakir Siddiquee, Sujata Nerle, Michal Stanczak, Michele Mayo, Lihui Xu, Karl Normington, Jeff Brown, Wei Yao, Carolyn Bertozzi, James Broderick, Heinz Läubli, Li Peng. A novel therapeutic modality of inhibiting the glyco-immune checkpoint axis to treat cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-109.

Role of the protein CagA from Helicobacter pylori in the modifications of the human B lymphocyte cytoskeleton

Abstract The cytotoxin-associated gen A (CagA) is an oncogenic virulence factor of Helicobacter pylori that is related with the development of gastric cancer and gastric MALToma: a B cell non-Hodgkin’s lymphoma. CagA has an important role in the development of cancer due to its promiscuous interaction with different signaling-mediators. The association of CagA with cytoskeleton regulators generates an effect that gives invasive characteristics to epithelial cells, thus explaining the evolution to late stages of the gastric carcinoma. Although the translocation of CagA has been demonstrated in B lymphocytes, its impact on MALToma development and its effects over B cell cytoskeleton have not been characterized. Human Raji B cells were transiently transfected to express an EGFP-tagged CagA fusion protein. In culture, these cells displayed altered morphological and adhesion properties when compared with control cells. Fibronectin induced-spreading and adhesion assays were performed to assess the changes observed. The transfected cells exhibited atypical size and shape descriptors, determined by analyses of confocal microscopy images. Concomitantly, the adhesion of CagA+ Raji cells was observed reduced. These phenotypes could be explained by the diminished basal levels of ezrin that we detect in the CagA+ cells, determined by Western blot, that contrast with a high expression of this actin-regulatory molecule in Raji control cells. Taken together, our results show that the expression of CagA produces important changes in B cell morphology as well as cell adhesion due to alteration in actin cytoskeleton regulation; changes that could explain an invasive and aggressive B lymphocyte profile.

The Balancing Act of Intrinsically Disordered Proteins: Enabling Functional Diversity while Minimizing Promiscuity

Intrinsically disordered proteins (IDPs) or regions (IDRs) perform diverse cellular functions, but are also prone to forming promiscuous and potentially deleterious interactions. We investigate the extent to which the properties of, and content in, IDRs have adapted to enable functional diversity while limiting interference from promiscuous interactions in the crowded cellular environment. Information on protein sequences, their predicted intrinsic disorder, and 3D structure contents is related to data on protein cellular concentrations, gene co-expression, and protein–protein interactions in the well-studied yeast Saccharomyces cerevisiae. Results reveal that both the protein IDR content and the frequency of “sticky” amino acids in IDRs (those more frequently involved in protein interfaces) decrease with increasing protein cellular concentration. This implies that the IDR content and the amino acid composition of IDRs experience negative selection as the protein concentration increases. In the S. cerevisiae protein–protein interaction network, the higher a protein's IDR content, the more frequently it interacts with IDR-containing partners, and the more functionally diverse the partners are. Employing a clustering analysis of Gene Ontology terms, we newly identify ~600 putative multifunctional proteins in S. cerevisiae. Strikingly, these proteins are enriched in IDRs and contribute significantly to all the observed trends. In particular, IDRs of multi-functional proteins feature more sticky amino acids than IDRs of their non-multifunctional counterparts, or the surfaces of structured yeast proteins. This property likely affords sufficient binding affinity for the functional interactions, commonly mediated by short IDR segments, thereby counterbalancing the loss in overall IDR conformational entropy upon binding.

Structural and Dynamical Order of a Disordered Protein: Molecular Insights into Conformational Switching of PAGE4 at the Systems Level.

Folded proteins show a high degree of structural order and undergo (fairly constrained) collective motions related to their functions. On the other hand, intrinsically disordered proteins (IDPs), while lacking a well-defined three-dimensional structure, do exhibit some structural and dynamical ordering, but are less constrained in their motions than folded proteins. The larger structural plasticity of IDPs emphasizes the importance of entropically driven motions. Many IDPs undergo function-related disorder-to-order transitions driven by their interaction with specific binding partners. As experimental techniques become more sensitive and become better integrated with computational simulations, we are beginning to see how the modest structural ordering and large amplitude collective motions of IDPs endow them with an ability to mediate multiple interactions with different partners in the cell. To illustrate these points, here, we use Prostate-associated gene 4 (PAGE4), an IDP implicated in prostate cancer (PCa) as an example. We first review our previous efforts using molecular dynamics simulations based on atomistic AWSEM to study the conformational dynamics of PAGE4 and how its motions change in its different physiologically relevant phosphorylated forms. Our simulations quantitatively reproduced experimental observations and revealed how structural and dynamical ordering are encoded in the sequence of PAGE4 and can be modulated by different extents of phosphorylation by the kinases HIPK1 and CLK2. This ordering is reflected in changing populations of certain secondary structural elements as well as in the regularity of its collective motions. These ordered features are directly correlated with the functional interactions of WT-PAGE4, HIPK1-PAGE4 and CLK2-PAGE4 with the AP-1 signaling axis. These interactions give rise to repeated transitions between (high HIPK1-PAGE4, low CLK2-PAGE4) and (low HIPK1-PAGE4, high CLK2-PAGE4) cell phenotypes, which possess differing sensitivities to the standard PCa therapies, such as androgen deprivation therapy (ADT). We argue that, although the structural plasticity of an IDP is important in promoting promiscuous interactions, the modulation of the structural ordering is important for sculpting its interactions so as to rewire with agility biomolecular interaction networks with significant functional consequences.