Structural Constraint of Osteopontin Facilitates Efficient Binding to CD44

Since the original description in 1996, the interaction between the cytokine osteopontin (OPN) and the homing receptor CD44 has been extensively studied in cancer, inflammation, bone remodeling, and various other conditions. Alternative splicing and extensive posttranslational modifications by both binding partners, as well as the possibility for lateral recruitment of additional membrane receptors or soluble co-ligands into a complex have left the exact molecular requirements for high-affinity OPN-CD44 binding unresolved. We now report that there is a moderate engagement between the unmodified molecules, which results in curved double-reciprocal plots for OPN titration, suggesting the existence of two binding sites or two binding conformations. Structural constraint of OPN, by immobilization or by addition of heparin, is required for its strong ligation of CD44. Prior literature provides evidence that heparin binding to OPN prompts the unfolding of a core element in the protein. This conformational adjustment may be essential for efficient CD44 interaction. The integrin α9β1 seems to compete with the OPN-CD44 engagement, while the integrin αVβ3 reflects additive binding, suggesting that the CD44 contact sites on OPN are downstream of the RGD motif but overlap with the SVVYGLR domain. Hyaluronate has no effect, placing the relevant domain on CD44 downstream of the N-terminus.

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Induction of acetylcholine receptor clustering by native polystyrene beads. Implication of an endogenous muscle-derived signalling system

Journal of Cell Science ◽

10.1242/jcs.102.3.543 ◽

1992 ◽

Vol 102 (3) ◽

pp. 543-555 ◽

Cited By ~ 2

Author(s):

L.P. Baker ◽

Q. Chen ◽

H.B. Peng

Keyword(s):

Growth Factor ◽

Cell Surface ◽

Acetylcholine Receptor ◽

Hot Spots ◽

Hot Spot ◽

Muscle Cells ◽

Membrane Receptors ◽

Heparin Binding ◽

Polystyrene Beads ◽

Myotomal Muscle

Aneural muscle cells in culture often form acetylcholine receptor (AChR) clusters, termed hot spots, which are similar to those found at the postsynaptic membrane both in structure and in molecular composition. Although hot spots form on both dorsal and ventral surfaces of the cell, the ventral ones are better characterized because of their association with sites of cell-substratum contact. To understand the stimuli and mechanisms involved in ventral hot spot formation, native, uncoated polystyrene beads were applied to cultured Xenopus myotomal muscle cells to create local membrane-substratum contacts. These beads were able to induce a postsynaptic-type development as evidenced by the clustering of AChRs and the development of a set of ultrastructural specializations, including membrane infoldings and a basement membrane. Whereas these native beads were effective in inducing clustering, beads coated with bovine serum albumin or treated with serum-containing medium were ineffective. Native beads were also capable of inducing clusters in serum-free medium, indicating that their effect was mediated by endogenous molecules that were locally presented by the beads, rather than by bead adsorption of components in the medium. Heparan sulfate proteoglycan (HSPG) is a major component of the muscle extracellular matrix and our previous study has shown that basic fibroblast growth factor (bFGF), a member of the heparin-binding growth factor (HBGF) family, and its receptor are present in Xenopus myotomal muscle during the period of synaptogenesis. Therefore, we tested the involvement of HBGF in bead induction. The results of this study show the following: (1) preincubation of cultures in heparin, which solubilizes matrix-bound HBGFs, suppressed the bead-induced AChR clustering. (2) Suramin, which interferes with the interaction between several growth factors and their receptors, also inhibited bead-induced clustering. (3) Tyrphostin, which blocks tyrosine kinase activity associated with a number of growth factor receptors, was also inhibitory to the bead effect. (4) The percentage of bead-induced AChR clusters was significantly enhanced by pretreating the cultures with bFGF prior to bead application. This exogenously applied bFGF could be largely removed by treatment of cultures with heparin, suggesting its association with HSPG at the cell surface. (5) An anti-bFGF neutralizing antiserum significantly reduced the efficacy of the bead stimulation. These data suggest that uncoated beads, which adhere to the cell surface and can mimic the cell-substratum interaction, effect a local presentation of HBGFs, such as bFGF, residing with the HSPG to their membrane receptors, thereby locally activating receptor-associated tyrosine kinases.(ABSTRACT TRUNCATED AT 400 WORDS)

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p53 modifications: exquisite decorations of the powerful guardian

Journal of Molecular Cell Biology ◽

10.1093/jmcb/mjz060 ◽

2019 ◽

Vol 11 (7) ◽

pp. 564-577 ◽

Cited By ~ 39

Author(s):

Yanqing Liu ◽

Omid Tavana ◽

Wei Gu

Keyword(s):

Cell Fate ◽

Posttranslational Modifications ◽

Stress Responses ◽

Developmental Disorders ◽

Adenosine Diphosphate ◽

Independent Action ◽

Cell Fate Decisions ◽

Viral Binding ◽

Binding Partners ◽

Related Pathway

AbstractThe last 40 years have witnessed how p53 rose from a viral binding protein to a central factor in both stress responses and tumor suppression. The exquisite regulation of p53 functions is of vital importance for cell fate decisions. Among the multiple layers of mechanisms controlling p53 function, posttranslational modifications (PTMs) represent an efficient and precise way. Major p53 PTMs include phosphorylation, ubiquitination, acetylation, and methylation. Meanwhile, other PTMs like sumoylation, neddylation, O-GlcNAcylation, adenosine diphosphate (ADP)-ribosylation, hydroxylation, and β-hydroxybutyrylation are also shown to play various roles in p53 regulation. By independent action or interaction, PTMs affect p53 stability, conformation, localization, and binding partners. Deregulation of the PTM-related pathway is among the major causes of p53-associated developmental disorders or diseases, especially in cancers. This review focuses on the roles of different p53 modification types and shows how these modifications are orchestrated to produce various outcomes by modulating p53 activities or targeted to treat different diseases caused by p53 dysregulation.

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Measuring stepwise binding of a thermally fluctuating particle to a cell membrane without labeling

10.1101/763680 ◽

2019 ◽

Author(s):

A. Rohrbach ◽

T. Meyer ◽

H. Kress

Keyword(s):

Cell Membrane ◽

Specific Binding ◽

Thermal Fluctuations ◽

Membrane Receptors ◽

Brownian Dynamic ◽

Label Free ◽

Binding Partners ◽

Label Free Detection ◽

A Cell ◽

Position And Orientation

ABSTRACTThermal motions enable a particle to probe the optimal interaction state when binding to a cell membrane. However, especially on the scale of microseconds and nanometers, position and orientation fluctuations are difficult to observe with common measurement technologies. Here we show that it is possible to detect single binding events of IgG-coated polystyrene beads, which are held in an optical trap nearby the cell membrane of a macrophage. Changes in the spatial and temporal thermal fluctuations of the particle were measured interferometrically and no fluorophore labelling was required. We demonstrate both by Brownian dynamic simulations and by experiments that sequential step-wise increases in the force constant of the bond between a bead and a cell of typically 20 pN / µm are clearly detectable. In addition, this technique provides estimates about binding rates and diffusion constants of membrane receptors. The simple approach of thermal noise tracking points out new strategies in understanding interactions between cells and particles, which are relevant for a large variety of processes including phagocytosis, drug delivery or the effects of small microplastics and particulates on cells.SIGNIFICANCEInteractions of cells with nearby particles, e.g. bacteria, viruses or synthetic material, is a very fundamental and complex process, often deciding about the cellular fate. The investigation of binding processes between particle and cell is typically investigated by fluorescence techniques, where fluorophores often hinder the molecular interaction of specific binding partners. Therefore, label-free detection or imaging techniques are essential, which are hardly available especially for live cell investigations. Molecular binding is based on thermal position and orientation fluctuations of the binding partners to find the best interaction state. Here, we present a label-free measurement technique that allows us to detect multiple stepwise binding events of molecules on an optically trapped particle close to the cell membrane.

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Dentine sialophosphoprotein signal in dentineogenesis and dentine regeneration

10.22203/ecm.v042a04 ◽

2021 ◽

Vol 42 ◽

pp. 43-62

Author(s):

MM Liu ◽

WT Li ◽

XM Xia ◽

F Wang ◽

M MacDougall ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Protein Kinase ◽

Dental Pulp ◽

Integrin Αvβ3 ◽

Cellular Membrane ◽

Membrane Receptors ◽

Mitogen Activated Protein Kinase ◽

Rgd Motif ◽

Mitogen Activated Protein ◽

Rgd Site

Dentineogenesis starts on odontoblasts, which synthesise and secrete non-collagenous proteins (NCPs) and collagen. When dentine is injured, dental pulp progenitors/mesenchymal stem cells (MSCs) can migrate to the injured area, differentiate into odontoblasts and facilitate formation of reactionary dentine. Dental pulp progenitor cell/MSC differentiation is controlled at given niches. Among dental NCPs, dentine sialophosphoprotein (DSPP) is a member of the small integrin-binding ligand N-linked glycoprotein (SIBLING) family, whose members share common biochemical characteristics such as an Arg-Gly-Asp (RGD) motif. DSPP expression is cell- and tissue-specific and highly seen in odontoblasts and dentine. DSPP mutations cause hereditary dentine diseases. DSPP is catalysed into dentine glycoprotein (DGP)/sialoprotein (DSP) and phosphoprotein (DPP) by proteolysis. DSP is further processed towards active molecules. DPP contains an RGD motif and abundant Ser-Asp/Asp-Ser repeat regions. DPP-RGD motif binds to integrin αVβ3 and activates intracellular signalling via mitogen-activated protein kinase (MAPK) and focal adhesion kinase (FAK)-ERK pathways. Unlike other SIBLING proteins, DPP lacks the RGD motif in some species. However, DPP Ser-Asp/Asp-Ser repeat regions bind to calcium-phosphate deposits and promote hydroxyapatite crystal growth and mineralisation via calmodulin-dependent protein kinase II (CaMKII) cascades. DSP lacks the RGD site but contains signal peptides. The tripeptides of the signal domains interact with cargo receptors within the endoplasmic reticulum that facilitate transport of DSPP from the endoplasmic reticulum to the extracellular matrix. Furthermore, the middle- and COOH-terminal regions of DSP bind to cellular membrane receptors, integrin β6 and occludin, inducing cell differentiation. The present review may shed light on DSPP roles during odontogenesis.

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Renal plasma membrane receptors for certain modified serum albumins Evidence for participation of a heparin receptor

Biochemical Journal ◽

10.1042/bj2390537 ◽

1986 ◽

Vol 239 (3) ◽

pp. 537-543 ◽

Cited By ~ 5

Author(s):

P N Ranganathan ◽

J L Mego

Keyword(s):

Plasma Membrane ◽

Plasma Membranes ◽

Dissociation Constants ◽

Chondroitin Sulphate ◽

Specific Binding ◽

Membrane Receptors ◽

Heparin Binding ◽

Serum Albumins ◽

Plasma Membrane Receptors ◽

Renal Plasma Membranes

Binding of formaldehyde-treated (f-alb), reduced-carboxymethylated (ac-alb) or reduced-acetamidated (am-alb) bovine serum albumins to purified rat renal plasma membranes was studied. Radioiodinated f-alb or ac-alb bound to kidney membranes while am-alb neither bound significantly nor competed with f-alb binding to kidney membranes. The binding was specific, saturable and heat- and proteinase-sensitive. Competition studies showed that f-alb and ac-alb sites may be the same on these membranes. To determine the role played by charge in binding, competition experiments with polyanions were performed. Polyanions such as nucleic acid or glycosaminoglycans were effective competitors of f-alb binding to cell membranes. Heparin was especially inhibitory, being several-fold more so than chondroitin sulphate. Completely reduced and carboxymethylated albumin was a better competitor than its partially modified counterpart. Furthermore, f-alb was a significant competitor of [35S]heparin binding to kidney membranes. Also, partially purified heparin receptor demonstrated specific binding of 125I-f-alb. These data suggest that a heparin receptor is responsible for binding and internalization of intravenously injected f-alb. A Scatchard plot revealed two classes of receptors with dissociation constants of 3.2 × 10(-6) M and 4.7 × 10(-5) M.

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Regulation of G Protein-Coupled Receptors by Ubiquitination

10.20944/preprints201704.0135.v1 ◽

2017 ◽

Cited By ~ 1

Author(s):

Kamila Skieterska ◽

Pieter Rondou ◽

Kathleen Van Craenenbroeck

Keyword(s):

G Protein ◽

Posttranslational Modifications ◽

Drug Targets ◽

Current Knowledge ◽

G Protein Coupled Receptors ◽

Membrane Receptors ◽

Deubiquitinating Enzymes ◽

Range Of Functions ◽

Beta Arrestins ◽

G Protein Coupled

G protein-coupled receptors (GPCRs) comprise the largest family of membrane receptors that control many cellular processes and consequently often serve as drug targets. These receptors undergo a strict regulation by mechanisms such as internalization and desensitization, which are strongly influenced by posttranslational modifications. Ubiquitination is a posttranslational modification with a broad range of functions that is currently gaining increased appreciation as a regulator of GPCR activity. The role of ubiquitination in directing GPCRs for lysosomal degradation has already been well-established. Furthermore, this modification can also play a role in targeting membrane and endoplasmic reticulum-associated receptors to the proteasome. Most recently, ubiquitination was also shown to be involved in GPCR signaling. In this review, we present current knowledge on the molecular basis of GPCR regulation by ubiquitination, and highlight the importance of E3 ubiquitin ligases, deubiquitinating enzymes and β-arrestins. Finally, we discuss classical and newly-discovered functions of ubiquitination in controlling GPCR activity.

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The Mycobacterial Heparin-Binding Hemagglutinin Is a Protective Antigen in the Mouse Aerosol Challenge Model of Tuberculosis

Infection and Immunity ◽

10.1128/iai.72.12.6799-6805.2004 ◽

2004 ◽

Vol 72 (12) ◽

pp. 6799-6805 ◽

Cited By ~ 60

Author(s):

Marcela Parra ◽

Thames Pickett ◽

Giovanni Delogu ◽

Veerabadran Dheenadhayalan ◽

Anne-Sophie Debrie ◽

...

Keyword(s):

Posttranslational Modifications ◽

Lipid A ◽

Protective Antigen ◽

Serum Antibody ◽

Passive Immunization ◽

Host Cells ◽

Interferon Response ◽

Heparin Binding ◽

Log Reduction ◽

Monophosphoryl Lipid A

ABSTRACT The heparin-binding hemagglutinin (HBHA) of Mycobacterium tuberculosis is a surface-expressed adhesin that can affect binding to host cells via a unique, methylated, carboxyl-terminal, lysine-, alanine-, and proline-rich repeat region. It has been implicated in extrapulmonary dissemination of M. tuberculosis from the lung following the initial infection of the host. To assess the vaccine potential of this protein, purified preparations of HBHA were emulsified in a dimethyldioctadecylammonium bromide-monophosphoryl lipid A adjuvant and tested for the ability to reduce M. tuberculosis infection in the mouse aerosol challenge model for tuberculosis. The HBHA-containing vaccine gave a ∼0.7-log reduction in CFU in both mouse lungs and spleens compared to adjuvant controls 28 days following challenge. Although a notable level of serum antibody to HBHA was elicited after three immunizations and the antibodies were able to bind to the surface of M. tuberculosis, passive immunization with monoclonal antibodies directed against HBHA did not protect in the challenge model. Compared to adjuvant controls, an elevated gamma interferon response was generated by splenic and lymph node-derived T cells from immunized mice in the presence of macrophages pulsed with purified HBHA or infected with live M. tuberculosis, suggesting that the effective immunity may be cell mediated. Efforts to construct effective recombinant HBHA vaccines in fast-growing Mycobacterium smegmatis have been unsuccessful so far, which indicates that distinctive posttranslational modifications present in the HBHA protein expressed by M. tuberculosis are critical for generating effective host immune responses. The vaccine studies described here demonstrate that HBHA is a promising new vaccine candidate for tuberculosis.

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Human Papillomavirus 31 Tyrosine 102 Regulates Interaction with E2 Binding Partners and Episomal Maintenance

Journal of Virology ◽

10.1128/jvi.00590-20 ◽

2020 ◽

Vol 94 (16) ◽

Author(s):

Timra Gilson ◽

Sara Culleton ◽

Fang Xie ◽

Marsha DeSmet ◽

Elliot J. Androphy

Keyword(s):

Posttranslational Modifications ◽

Phosphorylation Site ◽

Binding Partners ◽

Copy Numbers ◽

C Terminus ◽

Epithelial Cell Lines ◽

Matrix Fraction ◽

Episomal Maintenance ◽

Tyrosine Phosphorylation Site

ABSTRACT Several serine and threonine residues of the papillomavirus early E2 protein have been found to be phosphorylated. In contrast, only one E2 tyrosine phosphorylation site in BPV-1 (tyrosine 102) and one in HPV-16/31 (tyrosine 138) site have been characterized. Between BPV-1 and HPV-31 E2, 8 of the 11 tyrosines are conserved in the N-terminal domain, suggesting that phosphorylation of tyrosines has an essential role in E2 biology. In this study, we examine the effect of Y102 phosphorylation on HPV-31 E2 biology. Y102 proteins mutated either to the potential phospho-mimetic glutamic acid (Y102E) or to the nonphosphorylated homologue phenylalanine (Y102F) remain nuclear; however, Y102E is more associated with the nuclear matrix fraction. This is consistent with the inability of Y102E to bind TopBP1. Both BPV-1 and HPV-31 Y102E are similar in that neither binds the C terminus of Brd4, but in all other aspects the mutant behaves differently between the two families of papillomaviruses. BPV-1 Y102E was unable to bind E1 and did not replicate in a transient in vitro assay, while HPV-31 Y102E binds E1 and was able to replicate, albeit at lower levels than wild type. To examine the effect of E2 mutations under more native-like infection conditions, a neomycin-selectable marker was inserted into L1/L2 of the HPV-31 genome, creating HPV-31neo. This genome was maintained in every cell line tested for at least 50 days posttransfection/infection. Y102E in both transfection and infection conditions was unable to maintain high episome copy numbers in epithelial cell lines. IMPORTANCE Posttranslational modifications by phosphorylation can change protein activities, binding partners, or localization. Tyrosine 102 is conserved between delta papillomavirus BPV-1 and alpha papillomavirus HPV-31 E2. We characterized mutations of HPV-31 E2 for interactions with relevant cellular binding partners and replication in the context of the viral genome.

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Electroacupuncture Improves Pregnancy Outcomes in Rats with Thin Endometrium by Promoting the Expression of Pinopode-Related Molecules

BioMed Research International ◽

10.1155/2021/6658321 ◽

2021 ◽

Vol 2021 ◽

pp. 1-12

Author(s):

Jin Xi ◽

Jie Cheng ◽

Chun-chun Jin ◽

Jing-yu Liu ◽

Zhen-ru Shen ◽

...

Keyword(s):

Pregnancy Rate ◽

Endometrial Thickness ◽

Embryo Implantation ◽

Integrin Αvβ3 ◽

Endometrial Receptivity ◽

Control Group ◽

Heparin Binding ◽

Model Group ◽

Sprague Dawley ◽

Thin Endometrium

A thin endometrium affects the success of assisted reproduction due to low endometrial receptivity. Acupuncture improves endometrial receptivity and promotes the formation of pinopodes, the ultrastructure marker implantation window. However, the specific underlying mechanisms remain unclear. In this study, the efficacy of acupuncture treatment and its underlying mechanism were investigated by analyzing pregnancy rate, pinopode formation, and related molecular markers in thin endometrium model rats. Absolute ethanol (95%) was injected into the uteruses of female Sprague-Dawley rats to construct a thin endometrium model. In this model, acupuncture stimulation at EX-CA1, SP6, and CV4 ameliorated the pregnancy rate. Significantly increased embryo implantation, endometrial thickness, numbers of glands, and blood vessels were observed in the electroacupuncture (EA) group compared to the model group. The number of pinopodes in the EA group was abundant, with a shape similar to that of the control group. Additionally, significantly higher expression levels of pinopode-related markers, including integrin αvβ3, homeobox A10 (HOXA10), heparin-binding EGF-like growth factor (HBEGF), estrogen receptor alpha (ERα), and progesterone receptor (PR), were observed in the EA group than those in the model group. In conclusion, EA had a positive effect on the endometrial receptivity of thin endometrium model rats by improving pinopode formation through multiple molecular targets.

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The Many Faces of Rap1 GTPase

International Journal of Molecular Sciences ◽

10.3390/ijms19102848 ◽

2018 ◽

Vol 19 (10) ◽

pp. 2848 ◽

Cited By ~ 13

Author(s):

Anna Jaśkiewicz ◽

Beata Pająk ◽

Arkadiusz Orzechowski

Keyword(s):

Posttranslational Modifications ◽

Cell Types ◽

Membrane Receptors ◽

Nucleotide Exchange ◽

Guanine Nucleotide Exchange ◽

Nucleotide Exchange Factors ◽

Guanosine Triphosphatase ◽

The Many ◽

Plasma Membrane Receptors ◽

Cytoskeleton Rearrangements

This review addresses the issue of the numerous roles played by Rap1 GTPase (guanosine triphosphatase) in different cell types, in terms of both physiology and pathology. It is one among a myriad of small G proteins with endogenous GTP-hydrolyzing activity that is considerably stimulated by posttranslational modifications (geranylgeranylation) or guanine nucleotide exchange factors (GEFs), and inhibited by GTPase-activating proteins (GAPs). Rap1 is a ubiquitous protein that plays an essential role in the control of metabolic processes, such as signal transduction from plasma membrane receptors, cytoskeleton rearrangements necessary for cell division, intracellular and substratum adhesion, as well as cell motility, which is needed for extravasation or fusion. We present several examples of how Rap1 affects cells and organs, pointing to possible molecular manipulations that could have application in the therapy of several diseases.

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