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Sample records for integrin-binding matricellular protein

  1. Fibulin-5, an integrin-binding matricellular protein: its function in development and disease.

    PubMed

    Yanagisawa, Hiromi; Schluterman, Marie K; Brekken, Rolf A

    2009-12-01

    Interactions between the extracellular matrix (ECM) and cells are critical in embryonic development, tissue homeostasis, physiological remodeling, and tumorigenesis. Matricellular proteins, a group of ECM components, mediate cell-ECM interactions. One such molecule, Fibulin-5 is a 66-kDa glycoprotein secreted by various cell types, including vascular smooth muscle cells (SMCs), fibroblasts, and endothelial cells. Fibulin-5 contributes to the formation of elastic fibers by binding to structural components including tropoelastin and fibrillin-1, and to cross-linking enzymes, aiding elastic fiber assembly. Mice deficient in the fibulin-5 gene (Fbln5) exhibit systemic elastic fiber defects with manifestations of loose skin, tortuous aorta, emphysematous lung and genital prolapse. Although Fbln5 expression is down-regulated after birth, following the completion of elastic fiber formation, expression is reactivated upon tissue injury, affecting diverse cellular functions independent of its elastogenic function. Fibulin-5 contains an evolutionally conserved arginine-glycine-aspartic acid (RGD) motif in the N-terminal region, which mediates binding to a subset of integrins, including alpha5beta1, alphavbeta3, and alphavbeta5. Fibulin-5 enhances substrate attachment of endothelial cells, while inhibiting migration and proliferation in a cell type- and context-dependent manner. The antagonistic function of fibulin-5 in angiogenesis has been demonstrated in vitro and in vivo; fibulin-5 may block angiogenesis by inducing the anti-angiogenic molecule thrompospondin-1, by antagonizing VEGF(165)-mediated signaling, and/or by antagonizing fibronectin-mediated signaling through directly binding and blocking the alpha5beta1 fibronectin receptor. The overall effect of fibulin-5 on tumor growth depends on the balance between the inhibitory property of fibulin-5 on angiogenesis and the direct effect of fibulin-5 on proliferation and migration of tumor cells. However, the effect of

  2. Matricellular proteins and biomaterials

    PubMed Central

    Morris, Aaron H.; Kyriakides, Themis R.

    2014-01-01

    Biomaterials are essential to modern medicine as components of reconstructive implants, implantable sensors, and vehicles for localized drug delivery. Advances in biomaterials have led to progression from simply making implants that are nontoxic to making implants that are specifically designed to elicit particular functions within the host. The interaction of implants and the extracellular matrix during the foreign body response is a growing area of concern for the field of biomaterials, because it can lead to implant failure. Expression of matricellular proteins is modulated during the foreign body response and these proteins interact with biomaterials. The design of biomaterials to specifically alter the levels of matricellular proteins surrounding implants provides a new avenue for the design and fabrication of biomimetic biomaterials. PMID:24657843

  3. Matricellular proteins and biomaterials.

    PubMed

    Morris, Aaron H; Kyriakides, Themis R

    2014-07-01

    Biomaterials are essential to modern medicine as components of reconstructive implants, implantable sensors, and vehicles for localized drug delivery. Advances in biomaterials have led to progression from simply making implants that are nontoxic to making implants that are specifically designed to elicit particular functions within the host. The interaction of implants and the extracellular matrix during the foreign body response is a growing area of concern for the field of biomaterials, because it can lead to implant failure. Expression of matricellular proteins is modulated during the foreign body response and these proteins interact with biomaterials. The design of biomaterials to specifically alter the levels of matricellular proteins surrounding implants provides a new avenue for the design and fabrication of biomimetic biomaterials.

  4. Matricellular proteins in cardiac adaptation and disease.

    PubMed

    Frangogiannis, Nikolaos G

    2012-04-01

    The term matricellular proteins describes a family of structurally unrelated extracellular macromolecules that, unlike structural matrix proteins, do not play a primary role in tissue architecture, but are induced following injury and modulate cell-cell and cell-matrix interactions. When released to the matrix, matricellular proteins associate with growth factors, cytokines, and other bioactive effectors and bind to cell surface receptors transducing signaling cascades. Matricellular proteins are upregulated in the injured and remodeling heart and play an important role in regulation of inflammatory, reparative, fibrotic and angiogenic pathways. Thrombospondin (TSP)-1, -2, and -4 as well as tenascin-C and -X secreted protein acidic and rich in cysteine (SPARC), osteopontin, periostin, and members of the CCN family (including CCN1 and CCN2/connective tissue growth factor) are involved in a variety of cardiac pathophysiological conditions, including myocardial infarction, cardiac hypertrophy and fibrosis, aging-associated myocardial remodeling, myocarditis, diabetic cardiomyopathy, and valvular disease. This review discusses the properties and characteristics of the matricellular proteins and presents our current knowledge on their role in cardiac adaptation and disease. Understanding the role of matricellular proteins in myocardial pathophysiology and identification of the functional domains responsible for their actions may lead to design of peptides with therapeutic potential for patients with heart disease.

  5. Matricellular proteins: a sticky affair with cancers.

    PubMed

    Chong, Han Chung; Tan, Chek Kun; Huang, Royston-Luke; Tan, Nguan Soon

    2012-01-01

    The multistep process of metastasis is a major hallmark of cancer progression involving the cointeraction and coevolution of the tumor and its microenvironment. In the tumor microenvironment, tumor cells and the surrounding stromal cells aberrantly secrete matricellular proteins, which are a family of nonstructural proteins in the extracellular matrix (ECM) that exert regulatory roles via a variety of molecular mechanisms. Matricellular proteins provide signals that support tumorigenic activities characteristic of the metastastic cascade such as epithelial-to-mesenchymal (EMT) transition, angiogenesis, tumor cell motility, proliferation, invasion, evasion from immune surveillance, and survival of anoikis. Herein, we review the current understanding of the following matricellular proteins and highlight their pivotal and multifacted roles in metastatic progression: angiopoietin-like protein 4 (ANGPTL4), CCN family members cysteine-rich angiogenic inducer 61 (Cyr61/CCN1) and CCN6, osteopontin (OPN), secreted protein acidic and rich in cysteine (SPARC), tenascin C (TNC), and thrombospondin-1 and -2 (TSP1, TSP2). Insights into the signaling mechanisms resulting from the interaction of these matricellular proteins and their respective molecular partner(s), as well as their subsequent contribution to tumor metastasis, are discussed. In addition, emerging evidences of their promising potential as therapeutic options and/or targets in the treatment of cancer are also highlighted.

  6. Calcium- and integrin-binding protein 1 regulates megakaryocyte ploidy, adhesion, and migration

    PubMed Central

    Kostyak, John C.; Naik, Meghna U.

    2012-01-01

    Megakaryocytes are large, polyploid cells that produce platelets. We have previously reported that calcium- and integrin-binding protein 1 (CIB1) regulates endomitosis in Dami cells. To further characterize the role of CIB1 in megakaryopoiesis, we used a Cib1−/− mouse model. Cib1−/− mice have more platelets and BM megakaryocytes than wild-type (WT) controls (P < .05). Furthermore, subsequent analysis of megakaryocyte-CFU production revealed an increase with Cib1 deletion compared with WT (P < .05). In addition, BM from Cib1−/− mice, cultured with thrombopoietin (TPO) for 24 hours, produced more highly polyploid megakaryocytes than WT BM (P < .05). Subsequent analysis of TPO signaling revealed enhanced Akt and ERK1/2 phosphorylation, whereas FAKY925 phosphorylation was reduced in Cib1−/− megakaryocytes treated with TPO. Conversely, platelet recovery in Cib1−/− mice after platelet depletion was attenuated compared with WT (P < .05). This could be the result of impaired adhesion and migration, as adhesion to fibrinogen and fibronectin and migration toward an SDF-1α gradient were reduced in Cib1−/− megakaryocytes compared with WT (P < .05). In addition, Cib1−/− megakaryocytes formed fewer proplatelets compared with WT (P < .05), when plated on fibrinogen. These data suggest that CIB1 plays a dual role in megakaryopoiesis, initially by negatively regulating TPO signaling and later by augmenting proplatelet production. PMID:22128142

  7. Matricellular protein Cfl1 regulates cell differentiation.

    PubMed

    Tian, Xiuyun; Lin, Xiaorong

    2013-11-01

    Like higher eukaryotic cells in tissues, microbial cells in a community act in concert in response to environmental stimuli. They coordinate gene expression and their physiological and morphological states through intercellular communication mediated by matricellular signals. The adhesion protein Cfl1 was recently shown to be a matricellular signal in regulating morphogenesis and biofilm formation in the eukaryotic microbe Cryptococcus neoformans. Cfl1 is naturally highly expressed in the hyphal subpopulation during the mating colony development. Some Cfl1 proteins are cleaved and released to the ECM (extracellular matrix). The released exogenous Cfl1 activates Cryptococcus cells to express their endogenous Cfl1, to undergo filamentation, and to form structured biofilm colonies. In this study, we demonstrate that the N-terminal signal peptide and the novel conserved cysteine-rich SIGC domain at the C-terminus are critical for the adherence property and the signaling activity of this multifunctional protein. The investigation of this fungal matricellular signaling network involving Cfl1 and the master regulator of morphogenesis Znf2 provides a foundation to further elucidate intercellular communication in microbial development.

  8. Matricellular proteins: new molecular targets to prevent heart failure.

    PubMed

    Okamoto, Hiroshi; Imanaka-Yoshida, Kyoko

    2012-08-01

    Matricellular proteins are highly expressed in reparative responses to pressure and volume overload, ischemia, oxidative stress after myocardial injury, and modulate the inflammatory and fibrotic process in ventricular remodeling, which leads to cardiac dysfunction and eventually overt heart failure. Generally, matricellular proteins loosen strong adhesion of cardiomyocytes to extracellular matrix, which would help cells to move for rearrangement and allow inflammatory cells and capillary vessels to spread during tissue remodeling. Among matricellular proteins, osteopontin (OPN) and tenascin-C (TN-C) are de-adhesion proteins and upregulate the expression and activity of matrix metalloproteinases. These matricellular proteins could be key molecules to diagnose cardiac remodeling and also might be targets for the prevention of adverse ventricular remodeling. This review provides an overview of the role of matricellular proteins such as OPN and TN-C in cardiac function and remodeling, as determined by both in basic and in clinical studies.

  9. Purification and stable isotope labeling of the calcium- and integrin-binding protein 1 for structural and functional NMR studies.

    PubMed

    Huang, Hao; Vogel, Hans J

    2013-01-01

    The Calcium- and Integrin-Binding protein 1 (CIB1) has been identified as an important regulatory Ca(2+)-binding protein that is involved in various cellular functions. Nuclear Magnetic Resonance (NMR) spectroscopy provides a powerful approach to study the structure, dynamics, and interactions of CIB1 and related proteins. Multidimensional NMR spectroscopy combined with various selective isotope labeling strategies has proven to be successful in the structure determination of CIB1. Moreover, the same approach allowed the detection of conformational changes when the protein binds different metal ions, and it facilitated the study of the interaction of CIB1 with the cytoplasmic domain of the human integrin αIIb subunit. In this protocol, we describe the purification and isotope labeling strategies for productive NMR studies of CIB1. The same isotope labeling strategies can be implemented to study numerous related regulatory calcium-binding proteins.

  10. The role of matricellular proteins in glaucoma.

    PubMed

    Wallace, Deborah M; Murphy-Ullrich, Joanne E; Downs, J Crawford; O'Brien, Colm J

    2014-07-01

    Glaucoma is an optic neuropathy affecting approximately 60million people worldwide and is the second most common cause of irreversible blindness. Elevated intraocular pressure (IOP) is the main risk factor for developing glaucoma and is caused by impaired aqueous humor drainage through the trabecular meshwork (TM) and Schlemm's canal (SC). In primary open angle glaucoma (POAG), this elevation in IOP in turn leads to deformation at the optic nerve head (ONH) specifically at the lamina cribrosa (LC) region where there is also a deposition of extracellular matrix (ECM) molecules such as collagen and fibronectin. Matricellular proteins are non-structural secreted glycoproteins that help cells communicate with their surrounding ECM. This family of proteins includes connective tissue growth factor (CTGF), also known as CCN2, thrombospondins (TSPs), secreted protein acidic and rich in cysteine (SPARC), periostin, osteonectin, and Tenascin-C and -X and other ECM proteins. All members appear to play a role in fibrosis and increased ECM deposition. Most are widely expressed in tissues particularly in the TM and ONH and deficiency of TSP1 and SPARC have been shown to lower IOP in mouse models of glaucoma through enhanced outflow facility. The role of these proteins in glaucoma is emerging as some have an association with the pathophysiology of the TM and LC regions and might therefore be potential targets for therapeutic intervention in glaucoma.

  11. Novel regulators of cardiac inflammation: Matricellular proteins expand their repertoire.

    PubMed

    Rienks, Marieke; Papageorgiou, Anna-Pia

    2016-02-01

    More than 20years ago, Paul Bornstein coined the term matricellular protein to describe a group of secreted extracellular matrix proteins with de-adhesive properties. Though this is still true today, this family of proteins is vastly expanding with new emerging functions pushing the boundaries of this classic definition. In the heart, matricellular proteins have been extensively investigated in models of myocardial infarction, pressure overload, viral myocarditis and age-related cardiomyopathy with clear implications during cardiac fibrosis yet their involvement in regulating cardiac inflammation is less established. In this review, we describe our current understanding of the immune activation by damage- or pathogen-associated molecular pattern molecules during cardiac injury making a distinction between sterile versus non-sterile cardiac inflammation, and explain how matricellular proteins influence this crucial pathophysiological response in the heart.

  12. Matricellular proteins in development: perspectives from the Drosophila heart.

    PubMed

    Volk, T; Wang, S; Rotstein, B; Paululat, A

    2014-07-01

    The Drosophila model represents an attractive system in which to study the functional contribution of specific genes to organ development. Within the embryo, the heart tube serves as an informative developmental paradigm to analyze functional aspects of matricellular proteins. Here, we describe two essential extracellular matricellular proteins, Multiplexin (Mp) and Lonely heart (Loh). Each of these proteins contributes to the development and morphogenesis of the heart tube by regulating the activity/localization of essential extracellular proteins. Mp, which is secreted by heart cardioblasts and is specifically distributed in the lumen of the heart tube, binds to the signaling protein Slit, and facilitates its local signaling at the heart's luminal domain. Loh is an ADAMTS-like protein, which serves as an adapter protein to Pericardin (a collagen-like protein), promoting its specific localization at the abluminal domain of the heart tube. We also introduce the Drosophila orthologues of matricellular proteins present in mammals, including Thrombospondin, and SPARC, and discuss a possible role for Teneurins (Ten-A and Ten-M) in the heart. Understanding the role of these proteins provides a novel developmental perspective into the functional contribution of matricellular proteins to organ development.

  13. Matricellular proteins in the trabecular meshwork: review and update.

    PubMed

    Chatterjee, Ayan; Villarreal, Guadalupe; Rhee, Douglas J

    2014-08-01

    Abstract Primary open-angle glaucoma (POAG) is a leading cause of blindness worldwide, and intraocular pressure (IOP) is an important modifiable risk factor. IOP is a function of aqueous humor production and aqueous humor outflow, and it is thought that prolonged IOP elevation leads to optic nerve damage over time. Within the trabecular meshwork (TM), the eye's primary drainage system for aqueous humor, matricellular proteins generally allow cells to modulate their attachments with and alter the characteristics of their surrounding extracellular matrix (ECM). It is now well established that ECM turnover in the TM affects outflow facility, and matricellular proteins are emerging as significant players in IOP regulation. The formalized study of matricellular proteins in TM has gained increased attention. Secreted protein acidic and rich in cysteine (SPARC), myocilin, connective tissue growth factor (CTGF), and thrombospondin-1 and -2 (TSP-1 and -2) have been localized to the TM, and a growing body of evidence suggests that these matricellular proteins play an important role in IOP regulation and possibly the pathophysiology of POAG. As evidence continues to emerge, these proteins are now seen as potential therapeutic targets. Further study is warranted to assess their utility in treating glaucoma in humans.

  14. Integrated multiomics approach identifies calcium and integrin-binding protein-2 as a novel gene for pulse wave velocity

    PubMed Central

    Mangino, Massimo; Cecelja, Marina; Menni, Cristina; Tsai, Pei-Chien; Yuan, Wei; Small, Kerrin; Bell, Jordana; Mitchell, Gary F.; Chowienczyk, Phillip; Spector, Tim D.

    2016-01-01

    Background: Carotid-femoral pulse wave velocity (PWV) is an important measure of arterial stiffness, which is an independent predictor of cardiovascular morbidity and mortality. In this study, we used an integrated genetic, epigenetic and transcriptomics approach to uncover novel molecular mechanisms contributing to PWV. Methods and results: We measured PWV in 1505 healthy twins of European descendent. A genomewide association analysis was performed using standardized residual of the inverse of PWV. We identified one single-nucleotide polymorphism (rs7164338) in the calcium and integrin-binding protein-2 (CIB2) gene on chromosome 15q25.1 associated with PWV [β = −0.359, standard error (SE) = 0.07, P = 4.8 × 10–8]. The same variant was also associated with increased CIB2 expression in leucocytes (β = 0.034, SE = 0.008, P = 4.95 × 10–5) and skin (β = 0.072, SE = 0.01, P = 2.35 × 10–9) and with hypomethylation of the gene promoter (β = −0.899, SE = 0.098, P = 3.63 × 10–20). Conclusion: Our data indicate that reduced methylation of the CIB2 promoter in individuals carrying rs7164338 may lead to increased CIB2 expression. Given that CIB2 is thought to regulate intracellular calcium levels, an increase in protein levels may prevent the accumulation of serum calcium and phosphate, ultimately slowing down the process of vascular calcification. This study shows the power of integrating multiple omics to discover novel cardiovascular mechanisms. PMID:26378684

  15. Matricellular proteins: from homeostasis to inflammation, cancer, and metastasis.

    PubMed

    Chiodoni, Claudia; Colombo, Mario P; Sangaletti, Sabina

    2010-06-01

    The family of matricellular proteins comprises molecules with disparate biology. The main characteristic of matricellular proteins is to be expressed during tissue renewal and repair in order to "normalize" the tissue. Tumors are wound that do not heal, and tumor growth and metastasis can be viewed as a consequence of aberrant homeostasis, during which matricellular proteins are often upregulated. In the tumor microenvironment, they can be produced by both tumor cells and surrounding stromal cells, such as fibroblasts and macrophages. In this context, matricellular proteins can exert several functions that actively contribute to tumor progression. They may (a) regulate cellular adhesion and migration and extracellular matrix deposition, (b) control tumor infiltration by macrophages or other leukocytes, (c) affect tumor angiogenesis, (d) regulate TGFbeta and other growth factor receptor signals, (e) directly stimulate integrin receptors to transduce pro-survival or pro-migratory signals, and (f) regulate the wnt/beta-catenin pathways. Most of these functions contribute to settle a chronic low inflammatory state, whose involvement in tissue transformation and tumor progression is now established.

  16. Expression of integrin-binding protein Nischarin in metastatic breast cancer

    PubMed Central

    CHEN, JIE; FENG, WEI-LIANG; MO, WEN-JU; DING, XIAO-WEN; XIE, SHANG-NAO

    2015-01-01

    The present study aimed to investigate the expression of Nischarin protein in primary breast cancer (PBC), and to evaluate its role in tumor metastasis. Paired specimens of breast cancer tissues and adjacent normal tissues were surgically obtained from 60 patients with PBC at the Zhejiang Cancer Hospital (Hangzhou, China). Nischarin protein concentrations were determined by an ELISA assay. Breast cancer tissues exhibited a significantly lower concentration of Nischarin (5.86±3.19 ng/ml) compared with that of the adjacent noncancerous tissues (9.25±3.65 ng/ml; P<0.001). Furthermore, cancer tissue from patients with lymph node metastasis had significantly lower levels of Nischarin protein (4.69±2.40 ng/ml) than those of patients without lymph node metastasis (7.04±3.47 ng/ml; P=0.004). There was no significant difference in Nischarin protein expression levels between patients with grade I, II or III PBC (grade I, 5.44±3.57 ng/ml; grade II, 6.42±3.85 ng/ml and grade III, 5.10±1.18 ng/ml; P=0.765). The significant differences in the expression of Nischarin between: i) Cancer tissue and noncancerous tissue and ii) patients with and without lymph node metastasis, suggested that Nischarin may have a significant role in tumor occurrence and metastasis of breast cancer. Nischarin expression may therefore be used as a marker to predict the invasiveness and metastasis of PBC. PMID:25695373

  17. The matricellular protein CCN1 controls retinal angiogenesis by targeting VEGF, Src homology 2 domain phosphatase-1 and Notch signaling.

    PubMed

    Chintala, Hemabindu; Krupska, Izabela; Yan, Lulu; Lau, Lester; Grant, Maria; Chaqour, Brahim

    2015-07-01

    Physiological angiogenesis depends on the highly coordinated actions of multiple angiogenic regulators. CCN1 is a secreted cysteine-rich and integrin-binding matricellular protein required for proper cardiovascular development. However, our understanding of the cellular origins and activities of this molecule is incomplete. Here, we show that CCN1 is predominantly expressed in angiogenic endothelial cells (ECs) at the leading front of actively growing vessels in the mouse retina. Endothelial deletion of CCN1 in mice using a Cre-Lox system is associated with EC hyperplasia, loss of pericyte coverage and formation of dense retinal vascular networks lacking the normal hierarchical arrangement of arterioles, capillaries and venules. CCN1 is a product of an immediate-early gene that is transcriptionally induced in ECs in response to stimulation by vascular endothelial growth factor (VEGF). We found that CCN1 activity is integrated with VEGF receptor 2 (VEGF-R2) activation and downstream signaling pathways required for tubular network formation. CCN1-integrin binding increased the expression of and association between Src homology 2 domain-containing protein tyrosine phosphatase-1 (SHP-1) and VEGF-R2, which leads to rapid dephosphorylation of VEGF-R2 tyrosine, thus preventing EC hyperproliferation. Predictably, CCN1 further brings receptors/signaling molecules into proximity that are otherwise spatially separated. Furthermore, CCN1 induces integrin-dependent Notch activation in cultured ECs, and its targeted gene inactivation in vivo alters Notch-dependent vascular specification and remodeling, suggesting that functional levels of Notch signaling requires CCN1 activity. These data highlight novel functions of CCN1 as a naturally optimized molecule, fine-controlling key processes in physiological angiogenesis and safeguarding against aberrant angiogenic responses.

  18. Embracing the complexity of matricellular proteins: the functional and clinical significance of splice variation.

    PubMed

    Viloria, Katrina; Hill, Natasha J

    2016-05-01

    Matricellular proteins influence wide-ranging fundamental cellular processes including cell adhesion, migration, growth and differentiation. They achieve this both through interactions with cell surface receptors and regulation of the matrix environment. Many matricellular proteins are also associated with diverse clinical disorders including cancer and diabetes. Alternative splicing is a precisely regulated process that can produce multiple isoforms with variable functions from a single gene. To date, the expression of alternate transcripts for the matricellular family has been reported for only a handful of genes. Here we analyse the evidence for alternative splicing across the matricellular family including the secreted protein acidic and rich in cysteine (SPARC), thrombospondin, tenascin and CCN families. We find that matricellular proteins have double the average number of splice variants per gene, and discuss the types of domain affected by splicing in matricellular proteins. We also review the clinical significance of alternative splicing for three specific matricellular proteins that have been relatively well characterised: osteopontin (OPN), tenascin-C (TNC) and periostin. Embracing the complexity of matricellular splice variants will be important for understanding the sometimes contradictory function of these powerful regulatory proteins, and for their effective clinical application as biomarkers and therapeutic targets.

  19. Matricellular proteins as regulators of cancer metastasis to bone.

    PubMed

    Trotter, Timothy N; Yang, Yang

    2016-01-01

    Metastasis is the major cause of death in cancer patients, and a frequent site of metastasis for many cancers is the bone marrow. Therefore, understanding the mechanisms underlying the metastatic process is necessary for future prevention and treatment. The tumor microenvironment is now known to play a role in the metastatic cascade, both at the primary tumor and in metastatic sites, and includes both cellular and non-cellular components. The extracellular matrix (ECM) provides structural support and signaling cues to cells. One particular group of molecules associated with the ECM, known as matricellular proteins, modulate multiple aspects of tumor biology, including growth, migration, invasion, angiogenesis and metastasis. These proteins are also important for normal function in the bone by regulating bone formation and bone resorption. Recent studies have described a link between some of these proteins and metastasis of various tumors to the bone. The aim of this review is to summarize what is currently known about matricellular protein influence on bone metastasis. Particular attention to the contribution of both tumor cells and non-malignant cells in the bone has been given.

  20. Intravital Imaging of Vascular Transmigration by the Lyme Spirochete: Requirement for the Integrin Binding Residues of the B. burgdorferi P66 Protein

    PubMed Central

    Kumar, Devender; Ristow, Laura C.; Shi, Meiqing; Mukherjee, Priyanka; Caine, Jennifer A.; Lee, Woo-Yong; Kubes, Paul; Coburn, Jenifer; Chaconas, George

    2015-01-01

    Vascular extravasation, a key step in systemic infection by hematogenous microbial pathogens, is poorly understood, but has been postulated to encompass features similar to vascular transmigration by leukocytes. The Lyme disease spirochete can cause a variety of clinical manifestations, including arthritis, upon hematogenous dissemination. This pathogen encodes numerous surface adhesive proteins (adhesins) that may promote extravasation, but none have yet been implicated in this process. In this work we report the novel use of intravital microscopy of the peripheral knee vasculature to study transmigration of the Lyme spirochete in living Cd1d-/-mice. In the absence of iNKT cells, major immune modulators in the mouse joint, spirochetes that have extravasated into joint-proximal tissue remain in the local milieu and can be enumerated accurately. We show that BBK32, a fibronectin and glycosaminoglycan adhesin of B. burgdorferi involved in early steps of endothelial adhesion, is not required for extravasation from the peripheral knee vasculature. In contrast, almost no transmigration occurs in the absence of P66, an outer membrane protein that has porin and integrin adhesin functions. Importantly, P66 mutants specifically defective in integrin binding were incapable of promoting extravasation. P66 itself does not promote detectable microvascular interactions, suggesting that vascular adhesion of B. burgdorferi mediated by other adhesins, sets the stage for P66-integrin interactions leading to transmigration. Although integrin-binding proteins with diverse functions are encoded by a variety of bacterial pathogens, P66 is the first to have a documented and direct role in vascular transmigration. The emerging picture of vascular escape by the Lyme spirochete shows similarities, but distinct differences from leukocyte transmigration. PMID:26684456

  1. Roles of the Putative Integrin-Binding Motif of the Human Metapneumovirus Fusion (F) Protein in Cell-Cell Fusion, Viral Infectivity, and Pathogenesis

    PubMed Central

    Wei, Yongwei; Zhang, Yu; Cai, Hui; Mirza, Anne M.; Iorio, Ronald M.; Peeples, Mark E.; Niewiesk, Stefan

    2014-01-01

    ABSTRACT Human metapneumovirus (hMPV) is a relatively recently identified paramyxovirus that causes acute upper and lower respiratory tract infection. Entry of hMPV is unusual among the paramyxoviruses, in that fusion is accomplished by the fusion (F) protein without the attachment glycoprotein (G protein). It has been suggested that hMPV F protein utilizes integrin αvβ1 as a cellular receptor. Consistent with this, the F proteins of all known hMPV strains possess an integrin-binding motif (329RGD331). The role of this motif in viral entry, infectivity, and pathogenesis is poorly understood. Here, we show that α5β1 and αv integrins are essential for cell-cell fusion and hMPV infection. Mutational analysis found that residues R329 and G330 in the 329RGD331 motif are essential for cell-cell fusion, whereas mutations at D331 did not significantly impact fusion activity. Furthermore, fusion-defective RGD mutations were either lethal to the virus or resulted in recombinant hMPVs that had defects in viral replication in cell culture. In cotton rats, recombinant hMPV with the R329K mutation in the F protein (rhMPV-R329K) and rhMPV-D331A exhibited significant defects in viral replication in nasal turbinates and lungs. Importantly, inoculation of cotton rats with these mutants triggered a high level of neutralizing antibodies and protected against hMPV challenge. Taken together, our data indicate that (i) α5β1 and αv integrins are essential for cell-cell fusion and viral replication, (ii) the first two residues in the RGD motif are essential for fusion activity, and (iii) inhibition of the interaction of the integrin-RGD motif may serve as a new target to rationally attenuate hMPV for the development of live attenuated vaccines. IMPORTANCE Human metapneumovirus (hMPV) is one of the major causative agents of acute respiratory disease in humans. Currently, there is no vaccine or antiviral drug for hMPV. hMPV enters host cells via a unique mechanism, in that viral

  2. A novel splice variant of calcium and integrin-binding protein 1 mediates protein kinase D2-stimulated tumour growth by regulating angiogenesis.

    PubMed

    Armacki, M; Joodi, G; Nimmagadda, S C; de Kimpe, L; Pusapati, G V; Vandoninck, S; Van Lint, J; Illing, A; Seufferlein, T

    2014-02-27

    Protein kinase D2 (PKD2) is a member of the PKD family of serine/threonine kinases, a subfamily of the CAMK super-family. PKDs have a critical role in cell motility, migration and invasion of cancer cells. Expression of PKD isoforms is deregulated in various tumours and PKDs, in particular PKD2, have been implicated in the regulation of tumour angiogenesis. In order to further elucidate the role of PKD2 in tumours, we investigated the signalling context of this kinase by performing an extensive substrate screen by in vitro expression cloning (IVEC). We identified a novel splice variant of calcium and integrin-binding protein 1, termed CIB1a, as a potential substrate of PKD2. CIB1 is a widely expressed protein that has been implicated in angiogenesis, cell migration and proliferation, all important hallmarks of cancer, and CIB1a was found to be highly expressed in various cancer cell lines. We identify Ser(118) as the major PKD2 phosphorylation site in CIB1a and show that PKD2 interacts with CIB1a via its alanine and proline-rich domain. Furthermore, we confirm that CIB1a is indeed a substrate of PKD2 also in intact cells using a phosphorylation-specific antibody against CIB1a-Ser(118). Functional analysis of PKD2-mediated CIB1a phosphorylation revealed that on phosphorylation, CIB1a mediates tumour cell invasion, tumour growth and angiogenesis by mediating PKD-induced vascular endothelial growth factor secretion by the tumour cells. Thus, CIB1a is a novel mediator of PKD2-driven carcinogenesis and a potentially interesting therapeutic target. PMID:23503467

  3. Revisiting the matricellular concept

    PubMed Central

    Murphy-Ullrich, Joanne E.; Sage, E. Helene

    2015-01-01

    The concept of a matricellular protein was first proposed by Paul Bornstein in the mid-1990s to account for the non-lethal phenotypes of mice with inactivated genes encoding thrombospondin-1, tenascin-C, or SPARC. It was also recognized that these extracellular matrix proteins were primarily counter or de-adhesive. This review reappraises the matricellular concept after nearly two decades of continuous investigation. The expanded matricellular family as well as the diverse and often unexpected functions, cellular location, and interacting partners/receptors of matricellular proteins are considered. Development of therapeutic strategies that target matricellular proteins are discussed in the context of pathology and regenerative medicine. PMID:25064829

  4. Revisiting the matricellular concept.

    PubMed

    Murphy-Ullrich, Joanne E; Sage, E Helene

    2014-07-01

    The concept of a matricellular protein was first proposed by Paul Bornstein in the mid-1990s to account for the non-lethal phenotypes of mice with inactivated genes encoding thrombospondin-1, tenascin-C, or SPARC. It was also recognized that these extracellular matrix proteins were primarily counter or de-adhesive. This review reappraises the matricellular concept after nearly two decades of continuous investigation. The expanded matricellular family as well as the diverse and often unexpected functions, cellular location, and interacting partners/receptors of matricellular proteins are considered. Development of therapeutic strategies that target matricellular proteins are discussed in the context of pathology and regenerative medicine.

  5. A Novel C-Terminal CIB2 (Calcium and Integrin Binding Protein 2) Mutation Associated with Non-Syndromic Hearing Loss in a Hispanic Family.

    PubMed

    Patel, Kunjan; Giese, Arnaud P; Grossheim, J M; Hegde, Rashmi S; Hegde, Rashima S; Delio, Maria; Samanich, Joy; Riazuddin, Saima; Frolenkov, Gregory I; Cai, Jinlu; Ahmed, Zubair M; Morrow, Bernice E

    2015-01-01

    Hearing loss is a complex disorder caused by both genetic and environmental factors. Previously, mutations in CIB2 have been identified as a common cause of genetic hearing loss in Pakistani and Turkish populations. Here we report a novel (c.556C>T; p.(Arg186Trp)) transition mutation in the CIB2 gene identified through whole exome sequencing (WES) in a Caribbean Hispanic family with non-syndromic hearing loss. CIB2 belongs to the family of calcium-and integrin-binding (CIB) proteins. The carboxy-termini of CIB proteins are associated with calcium binding and intracellular signaling. The p.(Arg186Trp) mutation is localized within predicted type II PDZ binding ligand at the carboxy terminus. Our ex vivo studies revealed that the mutation did not alter the interactions of CIB2 with Whirlin, nor its targeting to the tips of hair cell stereocilia. However, we found that the mutation disrupts inhibition of ATP-induced Ca2+ responses by CIB2 in a heterologous expression system. Our findings support p.(Arg186Trp) mutation as a cause for hearing loss in this Hispanic family. In addition, it further highlights the necessity of the calcium binding property of CIB2 for normal hearing.

  6. Matricellular protein CCN3 mitigates abdominal aortic aneurysm

    PubMed Central

    Zhang, Chao; van der Voort, Dustin; Shi, Hong; Qing, Yulan; Hiraoka, Shuichi; Takemoto, Minoru; Yokote, Koutaro; Moxon, Joseph V.; Norman, Paul; Rittié, Laure; Atkins, G. Brandon; Gerson, Stanton L.; Shi, Guo-Ping; Golledge, Jonathan; Dong, Nianguo; Perbal, Bernard; Prosdocimo, Domenick A.

    2016-01-01

    Abdominal aortic aneurysm (AAA) is a major cause of morbidity and mortality; however, the mechanisms that are involved in disease initiation and progression are incompletely understood. Extracellular matrix proteins play an integral role in modulating vascular homeostasis in health and disease. Here, we determined that the expression of the matricellular protein CCN3 is strongly reduced in rodent AAA models, including angiotensin II–induced AAA and elastase perfusion–stimulated AAA. CCN3 levels were also reduced in human AAA biopsies compared with those in controls. In murine models of induced AAA, germline deletion of Ccn3 resulted in severe phenotypes characterized by elastin fragmentation, vessel dilation, vascular inflammation, dissection, heightened ROS generation, and smooth muscle cell loss. Conversely, overexpression of CCN3 mitigated both elastase- and angiotensin II–induced AAA formation in mice. BM transplantation experiments suggested that the AAA phenotype of CCN3-deficient mice is intrinsic to the vasculature, as AAA was not exacerbated in WT animals that received CCN3-deficient BM and WT BM did not reduce AAA severity in CCN3-deficient mice. Genetic and pharmacological approaches implicated the ERK1/2 pathway as a critical regulator of CCN3-dependent AAA development. Together, these results demonstrate that CCN3 is a nodal regulator in AAA biology and identify CCN3 as a potential therapeutic target for vascular disease. PMID:26974158

  7. Matricellular protein CCN3 mitigates abdominal aortic aneurysm.

    PubMed

    Zhang, Chao; van der Voort, Dustin; Shi, Hong; Zhang, Rongli; Qing, Yulan; Hiraoka, Shuichi; Takemoto, Minoru; Yokote, Koutaro; Moxon, Joseph V; Norman, Paul; Rittié, Laure; Kuivaniemi, Helena; Atkins, G Brandon; Gerson, Stanton L; Shi, Guo-Ping; Golledge, Jonathan; Dong, Nianguo; Perbal, Bernard; Prosdocimo, Domenick A; Lin, Zhiyong

    2016-04-01

    Abdominal aortic aneurysm (AAA) is a major cause of morbidity and mortality; however, the mechanisms that are involved in disease initiation and progression are incompletely understood. Extracellular matrix proteins play an integral role in modulating vascular homeostasis in health and disease. Here, we determined that the expression of the matricellular protein CCN3 is strongly reduced in rodent AAA models, including angiotensin II-induced AAA and elastase perfusion-stimulated AAA. CCN3 levels were also reduced in human AAA biopsies compared with those in controls. In murine models of induced AAA, germline deletion of Ccn3 resulted in severe phenotypes characterized by elastin fragmentation, vessel dilation, vascular inflammation, dissection, heightened ROS generation, and smooth muscle cell loss. Conversely, overexpression of CCN3 mitigated both elastase- and angiotensin II-induced AAA formation in mice. BM transplantation experiments suggested that the AAA phenotype of CCN3-deficient mice is intrinsic to the vasculature, as AAA was not exacerbated in WT animals that received CCN3-deficient BM and WT BM did not reduce AAA severity in CCN3-deficient mice. Genetic and pharmacological approaches implicated the ERK1/2 pathway as a critical regulator of CCN3-dependent AAA development. Together, these results demonstrate that CCN3 is a nodal regulator in AAA biology and identify CCN3 as a potential therapeutic target for vascular disease.

  8. Evaluation of matricellular proteins in systemic and local immune response to Mycobacterium tuberculosis infection.

    PubMed

    Hasibuan, Fakhrial Mirwan; Shiratori, Beata; Senoputra, Muhammad Andrian; Chagan-Yasutan, Haorile; Koesoemadinata, Raspati Cundarani; Apriani, Lika; Takahashi, Yayoi; Niki, Toshiro; Alisjahbana, Bachti; Hattori, Toshio

    2015-10-01

    Matricellular proteins such as osteopontin (OPN), galectin-9 (Gal-9), and tenascin-C (TN-C) are expressed not only under normal physiological conditions, but also during infection, inflammation and tumorigenesis. Plasma concentrations of matricellular proteins were studied to determine their diagnostic value as potential markers of tuberculosis (TB) activity. It was found that concentrations of OPN and TN-C were higher in patients with active TB than in healthy controls and individuals with latent infection. Moreover, LTBI patients had higher concentrations of OPN than did healthy controls. Gal-9 concentrations did not differ significantly between groups. Concentrations of matricellular proteins were higher in pleural fluid than in the plasma of patients with TB. Expression of matricellular proteins was also investigated in TB granulomas and other granulomatous diseases. Positive OPN and Gal-9 staining was observed in TB and sarcoidosis granulomas, but not in Crohn disease granulomas. The fibrotic ring around granulomas stained positive for TN-C in TB and sarcoidosis, but not in Crohn disease. Of the three matricellular proteins studied, OPN and TN-C may serve as reliable plasma markers for monitoring TB activity, whereas Gal-9 seems to be expressed more at the site of infection than in the systemic circulation.

  9. A matricellular protein and EGF-like repeat signalling in the social amoebozoan Dictyostelium discoideum.

    PubMed

    Huber, Robert J; O'Day, Danton H

    2012-12-01

    Matricellular proteins interact with the extracellular matrix (ECM) and modulate cellular processes by binding to cell surface receptors and initiating intracellular signal transduction. Their association with the ECM and the ability of some members of this protein family to regulate cell motility have opened up new avenues of research to investigate their functions in normal and diseased cells. In this review, we summarize the research on CyrA, an ECM calmodulin-binding protein in Dictyostelium. CyrA is proteolytically cleaved into smaller EGF-like (EGFL) repeat containing cleavage products during development. The first EGFL repeat of CyrA binds to the cell surface and activates a novel signalling pathway that modulates cell motility in this model organism. The similarity of CyrA to the most well-characterized matricellular proteins in mammals allows it to be designated as the first matricellular protein identified in Dictyostelium. PMID:22782112

  10. Matricellular proteins in drug delivery: Therapeutic targets, active agents, and therapeutic localization.

    PubMed

    Sawyer, Andrew J; Kyriakides, Themis R

    2016-02-01

    Extracellular matrix is composed of a complex array of molecules that together provide structural and functional support to cells. These properties are mainly mediated by the activity of collagenous and elastic fibers, proteoglycans, and proteins such as fibronectin and laminin. ECM composition is tissue-specific and could include matricellular proteins whose primary role is to modulate cell-matrix interactions. In adults, matricellular proteins are primarily expressed during injury, inflammation and disease. Particularly, they are closely associated with the progression and prognosis of cardiovascular and fibrotic diseases, and cancer. This review aims to provide an overview of the potential use of matricellular proteins in drug delivery including the generation of therapeutic agents based on the properties and structures of these proteins as well as their utility as biomarkers for specific diseases.

  11. A matricellular protein and EGF-like repeat signalling in the social amoebozoan Dictyostelium discoideum.

    PubMed

    Huber, Robert J; O'Day, Danton H

    2012-12-01

    Matricellular proteins interact with the extracellular matrix (ECM) and modulate cellular processes by binding to cell surface receptors and initiating intracellular signal transduction. Their association with the ECM and the ability of some members of this protein family to regulate cell motility have opened up new avenues of research to investigate their functions in normal and diseased cells. In this review, we summarize the research on CyrA, an ECM calmodulin-binding protein in Dictyostelium. CyrA is proteolytically cleaved into smaller EGF-like (EGFL) repeat containing cleavage products during development. The first EGFL repeat of CyrA binds to the cell surface and activates a novel signalling pathway that modulates cell motility in this model organism. The similarity of CyrA to the most well-characterized matricellular proteins in mammals allows it to be designated as the first matricellular protein identified in Dictyostelium.

  12. The Role of Matricellular Proteins in Brain Edema after Subarachnoid Hemorrhage.

    PubMed

    Suzuki, Hidenori; Fujimoto, Masashi; Shiba, Masato; Kawakita, Fumihiro; Liu, Lei; Ichikawa, Naoki; Kanamaru, Kenji; Imanaka-Yoshida, Kyoko; Yoshida, Toshimichi

    2016-01-01

    Accumulated evidence suggests that blood-brain barrier disruption or brain edema is an important pathologic manifestation for poor outcome after aneurysmal subarachnoid hemorrhage. Many molecules may be involved, acting simultaneously or at different stages during blood-brain barrier disruption via multiple independent or interconnected signaling pathways. Matricellular protein is a class of nonstructural, secreted, and multifunctional extracellular matrix proteins, which potentially mediates brain edema formation. This study reviews the role of osteopontin and tenascin-C, representatives of matricellular proteins, in the context of brain edema formation after subarachnoid hemorrhage in both clinical and experimental settings.

  13. The integrin-binding motif RGDS induces protein tyrosine phosphorylation without activation in Bufo arenarum (Amphibia) oocytes.

    PubMed

    Mouguelar, Valeria S; Cabada, Marcelo O; Coux, Gabriela

    2011-05-01

    Integrins are cell adhesion molecules that are thought to be involved in sperm-oocyte interaction. Nevertheless, their function in mammalian fertilization is still controversial, as different species behave differently. In amphibians, their role is mainly supported by Xenopus laevis studies, where RGDS peptide induces oocyte activation. We recently provided evidence suggesting the presence and involvement of integrins in the interaction of the oocyte plasma membrane (PM) with sperm in the amphibian Bufo arenarum. In order to understand the role of integrin homologs in oocytes and their possible contribution to egg activation mechanisms, we examined the presence of integrin subunits and the effect of RGDS peptide on oocytes and during fertilization. Western blot studies detected integrin subunits α5, αV and β1 in oocytes. In sperm, we could detect only the αV integrin subunit. We found that RGDS peptide was unable to elicit egg activation or MAPK dephosphorylation, but can induce reversible inhibition of fertilization. A similar partial inhibition was produced by an anti-β1 integrin antibody. Using an anti-phosphotyrosine antibody we found major changes in phosphotyrosine-containing proteins in egg extracts minutes after fertilization. Cytosol and PMs isolated from oocytes and fertilized eggs showed additional fertilization-induced phosphorylated proteins. Some of these were also present in cytosol and PMs from RGDS-treated oocytes (partially mimicking fertilization). These findings suggest that B. arenarum fertilization involves integrins (e.g. β1 subunit) as adhesion proteins. Our data support the view that RGDS-binding receptors may function as signaling receptors in B. arenarum oocytes, but integrin engagement by RGDS is not sufficient for oocyte activation. PMID:21339287

  14. Vitronectin in vascular context: facets of a multitalented matricellular protein.

    PubMed

    Preissner, Klaus T; Reuning, Ute

    2011-06-01

    Vitronectin is an abundant adhesive glycoprotein in blood plasma and is found associated with different extracellular matrix sites, the vessel wall, and tumor cells, particularly upon tissue remodeling, injury/repair, or under disease conditions. Plasma vitronectin is a structurally labile molecule that may be converted into a multimeric/multivalent form by interaction with various (hemostatic) factors or through surface binding. Several distinct binding domains along the vitronectin sequence for integrin-type cell adhesion receptors, for urokinase receptor or proteoglycans as well as for growth factors, endow vascular matrix- or fibrin-associated vitronectin with differentiated cell attachment and aggregatory properties. These were found to be relevant for modulation of the cell-matrix interface in angiogenesis, hemostasis and thrombus formation, or wound repair, respectively. Other vitronectin ligands include plasminogen activator inhibitor (PAI)-1 or high molecular weight kininogen that confer strong antiadhesive functions upon integrin- or urokinase receptor-mediated cell interactions with vitronectin. Together, vitronectin acts as a potent matricellular factor, coordinating cell migration with pericellular proteolysis and growth factor signaling at sites of tissue remodeling or in tumors. Structure-function studies of such vitronectin-related ligands and receptors lead to the characterization of their mode of action, also stimulating the search for new antagonists in tumor angiogenesis, platelet aggregation, or atherosclerosis. This review focuses on new developments in vitronectin biology, with particular emphasis on regulatory mechanisms of the protein in the context of cell adhesion/migration/proliferation and cell-dependent proteolysis, relevant for our understanding of hemostasis, thrombosis, tissue repair, and vascular diseases.

  15. Targeting the extracellular matrix: matricellular proteins regulate cell-extracellular matrix communication within distinct niches of the intervertebral disc.

    PubMed

    Bedore, Jake; Leask, Andrew; Séguin, Cheryle A

    2014-07-01

    The so-called "matricellular" proteins have recently emerged as important regulators of cell-extracellular matrix (ECM) interactions. These proteins modulate a variety of cell functions through a range of interactions with cell-surface receptors, hormones, proteases and structural components of the ECM. As such, matricellular proteins are crucial regulators of cell phenotype, and consequently tissue function. The distinct cell types and microenvironments that together form the IVD provide an excellent paradigm to study how matricellular proteins mediate communication within and between adjacent tissue types. In recent years, the role of several matricellular proteins in the intervertebral disc has been explored in vivo using mutant mouse models in which the expression of target matricellular proteins was deleted from either one or all compartments of the intervertebral disc. The current review outlines what is presently known about the roles of the matricellular proteins belonging to the CCN family, SPARC (Secreted Protein, Acidic, and Rich in Cysteine), and thrombospondin (TSP) 2 in regulating intervertebral disc cell-ECM interactions, ECM synthesis and disc tissue homeostasis using genetically modified mouse models. Furthermore, we provide a brief overview of recent preliminary studies of other matricellular proteins including, periostin (POSTN) and tenascin (TN). Each specific tissue type of the IVD contains a different matricellular protein signature, which varies based on the specific stage of development, maturity or disease. A growing body of direct genetic evidence links IVD development, maintenance and repair to the coordinate interaction of matricellular proteins within their respective niches and suggests that several of these signaling modulators hold promise in the development of diagnostics and/or therapeutics targeting intervertebral disc aging and/or degeneration.

  16. Matricellular proteins: priming the tumour microenvironment for cancer development and metastasis.

    PubMed

    Wong, G S; Rustgi, A K

    2013-03-01

    Matricellular proteins have been classified as a family of non-structural matrix proteins capable of modulating a variety of biological processes within the extracellular matrix (ECM). These proteins are expressed dynamically and their cellular functions are highly dependent upon cues from the local environment. Recent studies have shown an increasing appreciation of the key roles these ECM proteins play within the tumour microenvironment. Induced by either tumour cells or tumour stromal components, matricellular proteins initiate downstream signalling events that lead to proliferation, invasion, matrix remodelling and dissemination to pre-metastatic niches in other organs. In this review, we summarise and discuss the current knowledge of the diverse roles these proteins play within the microenvironment that influences tumour progression and potential for future therapies targeting the tumour microenvironment.

  17. The de-adhesive activity of matricellular proteins: is intermediate cell adhesion an adaptive state?

    PubMed

    Murphy-Ullrich, J E

    2001-04-01

    The process of cellular de-adhesion is potentially important for the ability of a cell to participate in morphogenesis and to respond to injurious stimuli. Cellular de-adhesion is induced by the highly regulated matricellular proteins TSP1 and 2, tenascin-C, and SPARC. These proteins induce a rapid transition to an intermediate state of adhesiveness characterized by loss of actin-containing stress fibers and restructuring of the focal adhesion plaque that includes loss of vinculin and alpha-actinin, but not of talin or integrin. This process involves intracellular signaling mediators, which are engaged in response to matrix protein-receptor interactions. Each of these proteins employs different receptors and signaling pathways to achieve this common morphologic endpoint. What is the function of this intermediate adhesive state and what is the physiologic significance of this action of the matricellular proteins? Given that matricellular proteins are expressed in response to injury and during development, one can speculate that the intermediate adhesive state is an adaptive condition that facilitates expression of specific genes that are involved in repair and adaptation. Since cell shape is maintained in weakly adherent cells, this state might induce survival signals to prevent apoptosis due to loss of strong cell adhesion, but yet allow for cell locomotion. The three matricellular proteins considered here might each preferentially facilitate one or more aspects of this adaptive response rather than all of these equally. Currently, we have only preliminary data to support the specific ideas proposed in this article. It will be interesting in the next several years to continue to elucidate the biological roles of the intermediate adhesive state induced by these matricellular proteins. and focal adhesions in a cell that nevertheless maintains a spread, extended morphology and integrin clustering. TSP1, tenascin-C, and SPARC induce the intermediate adhesive state, as

  18. Matricellular proteins of the Cyr61/CTGF/NOV (CCN) family and the nervous system

    PubMed Central

    Malik, Anna R.; Liszewska, Ewa; Jaworski, Jacek

    2015-01-01

    Matricellular proteins are secreted proteins that exist at the border of cells and the extracellular matrix (ECM). However, instead of playing a role in structural integrity of the ECM, these proteins, that act as modulators of various surface receptors, have a regulatory function and instruct a multitude of cellular responses. Among matricellular proteins are members of the Cyr61/CTGF/NOV (CCN) protein family. These proteins exert their activity by binding directly to integrins and heparan sulfate proteoglycans and activating multiple intracellular signaling pathways. CCN proteins also influence the activity of growth factors and cytokines and integrate their activity with integrin signaling. At the cellular level, CCN proteins regulate gene expression and cell survival, proliferation, differentiation, senescence, adhesion, and migration. To date, CCN proteins have been extensively studied in the context of osteo- and chondrogenesis, angiogenesis, and carcinogenesis, but the expression of these proteins is also observed in a variety of tissues. The role of CCN proteins in the nervous system has not been systematically studied or described. Thus, the major aim of this review is to introduce the CCN protein family to the neuroscience community. We first discuss the structure, interactions, and cellular functions of CCN proteins and then provide a detailed review of the available data on the neuronal expression and contribution of CCN proteins to nervous system development, function, and pathology. PMID:26157362

  19. Matricellular proteins of the Cyr61/CTGF/NOV (CCN) family and the nervous system.

    PubMed

    Malik, Anna R; Liszewska, Ewa; Jaworski, Jacek

    2015-01-01

    Matricellular proteins are secreted proteins that exist at the border of cells and the extracellular matrix (ECM). However, instead of playing a role in structural integrity of the ECM, these proteins, that act as modulators of various surface receptors, have a regulatory function and instruct a multitude of cellular responses. Among matricellular proteins are members of the Cyr61/CTGF/NOV (CCN) protein family. These proteins exert their activity by binding directly to integrins and heparan sulfate proteoglycans and activating multiple intracellular signaling pathways. CCN proteins also influence the activity of growth factors and cytokines and integrate their activity with integrin signaling. At the cellular level, CCN proteins regulate gene expression and cell survival, proliferation, differentiation, senescence, adhesion, and migration. To date, CCN proteins have been extensively studied in the context of osteo- and chondrogenesis, angiogenesis, and carcinogenesis, but the expression of these proteins is also observed in a variety of tissues. The role of CCN proteins in the nervous system has not been systematically studied or described. Thus, the major aim of this review is to introduce the CCN protein family to the neuroscience community. We first discuss the structure, interactions, and cellular functions of CCN proteins and then provide a detailed review of the available data on the neuronal expression and contribution of CCN proteins to nervous system development, function, and pathology.

  20. Astrocyte-secreted matricellular proteins in CNS remodelling during development and disease.

    PubMed

    Jones, Emma V; Bouvier, David S

    2014-01-01

    Matricellular proteins are secreted, nonstructural proteins that regulate the extracellular matrix (ECM) and interactions between cells through modulation of growth factor signaling, cell adhesion, migration, and proliferation. Despite being well described in the context of nonneuronal tissues, recent studies have revealed that these molecules may also play instrumental roles in central nervous system (CNS) development and diseases. In this minireview, we discuss the matricellular protein families SPARC (secreted protein acidic and rich in cysteine), Hevin/SC1 (SPARC-like 1), TN-C (Tenascin C), TSP (Thrombospondin), and CCN (CYR61/CTGF/NOV), which are secreted by astrocytes during development. These proteins exhibit a reduced expression in adult CNS but are upregulated in reactive astrocytes following injury or disease, where they are well placed to modulate the repair processes such as tissue remodeling, axon regeneration, glial scar formation, angiogenesis, and rewiring of neural circuitry. Conversely, their reexpression in reactive astrocytes may also lead to detrimental effects and promote the progression of neurodegenerative diseases.

  1. Mapping the Effect of Gly Mutations in Collagen on α2β1 Integrin Binding*

    PubMed Central

    Yigit, Sezin; Yu, Hongtao; An, Bo; Hamaia, Samir; Farndale, Richard W.; Kaplan, David L.; Lin, Yu-Shan; Brodsky, Barbara

    2016-01-01

    The replacement of one Gly in the essential repeating tripeptide sequence of the type I collagen triple helix results in the dominant hereditary bone disorder osteogenesis imperfecta. The mechanism leading to pathology likely involves misfolding and autophagy, although it has been hypothesized that some mutations interfere with known collagen interactions. Here, the effect of Gly replacements within and nearby the integrin binding GFPGER sequence was investigated using a recombinant bacterial collagen system. When a six-triplet human type I collagen sequence containing GFPGER was introduced into a bacterial collagen-like protein, this chimeric protein bound to integrin. Constructs with Gly to Ser substitutions within and nearby the inserted human sequence still formed a trypsin-resistant triple helix, suggesting a small local conformational perturbation. Gly to Ser mutations within the two Gly residues in the essential GFPGER sequence prevented integrin binding and cell attachment as predicted from molecular dynamics studies of the complex. Replacement of Gly residues C-terminal to GFPGER did not affect integrin binding. In contrast, Gly replacements N-terminal to the GFPGER sequence, up to four triplets away, decreased integrin binding and cell adhesion. This pattern suggests either an involvement of the triplets N-terminal to GFPGER in initial binding or a propagation of the perturbation of the triple helix C-terminal to a mutation site. The asymmetry in biological consequences relative to the mutation site may relate to the observed pattern of osteogenesis imperfecta mutations near the integrin binding site. PMID:27432884

  2. Oncostatin M is a novel inhibitor of TGF-β1-induced matricellular protein expression.

    PubMed

    Sarközi, Rita; Hauser, Christine; Noppert, Susie-Jane; Kronbichler, Andreas; Pirklbauer, Markus; Haller, Viktoria Maria; Grillari, Johannes; Grillari-Voglauer, Regina; Mayer, Gert; Schramek, Herbert

    2011-11-01

    Matricellular proteins in the kidney have been associated with the development of tubulointerstitial fibrogenesis and the progression of renal disease. This study investigated potential antifibrotic effects of the cytokine oncostatin M (OSM) in human proximal tubule cells (PTC), particularly with regard to inhibition of profibrotic events initiated by TGF-β1. In human PTC, OSM diminished transforming growth factor (TGF)-β1-induced expression of the transcriptional epithelial-mesenchymal transition mediator FoxC2. Furthermore, exposure to OSM attenuated basal and TGF-β1-induced expression of the matricellular proteins SPARC, TSP-1, TNC, and CTGF regardless of the sequence of ligand administration. OSM was shown to result in rapid and sustained phosphorylation of both Stat1 and Stat3 and also in transient phosphorylation of Smad2/3 in contrast to TGF-β1, which demonstrated a gradually building phosphorylation of Smad2/3 and a brief phosphorylation of Smad1/5/8. Utilizing receptor-blocking molecules, we found the inhibitory effect of OSM on TGF-β1-induced CTGF mRNA expression occurs independently of Smad2/3 signaling and present evidence that this effect may be partially driven by OSM receptor-mediated Stat1 and/or Stat3 signaling pathways, thereby providing a mechanism whereby OSM can contribute to tubulointerstitial protection.

  3. Periostin, a multifunctional matricellular protein in inflammatory and tumor microenvironments.

    PubMed

    Liu, Allan Yi; Zheng, Hong; Ouyang, Gaoliang

    2014-07-01

    The behavior and fate of cells in tissues largely rely upon their cross-talk with the tissue microenvironment including neighboring cells, the extracellular matrix (ECM), and soluble cues from the local and systemic environments. Dysregulation of tissue microenvironment can drive various inflammatory diseases and tumors. The ECM is a crucial component of tissue microenvironment. ECM proteins can not only modulate tissue microenvironment but also regulate the behavior of surrounding cells and the homeostasis of tissues. As a nonstructural ECM protein, periostin is generally present at low levels in most adult tissues; however, periostin is often highly expressed at sites of injury or inflammation and in tumors within adult organisms. Current evidence demonstrates that periostin actively contributes to tissue injury, inflammation, fibrosis and tumor progression. Here, we summarize the roles of periostin in inflammatory and tumor microenvironments.

  4. Critical role of the matricellular protein SPARC in mediating erythroid progenitor cell development in zebrafish.

    PubMed

    Ceinos, Rosa M; Torres-Nuñez, Eva; Chamorro, Ruben; Novoa, Beatriz; Figueras, Antonio; Ruane, Neil M; Rotllant, Josep

    2013-01-01

    Sparc (osteonectin) is a multifunctional matricellular glycoprotein expressed by many differentiated cells. Members of this family mediate cell-matrix interactions rather than acting as structural components of the extracellular matrix (ECM); therefore, they can influence many remodelling events, including haematopoiesis. We have investigated the role of sparc in embryonic haematopoiesis using a morpholino antisense oligonucleotide-based knockdown approach. Knockdown of sparc function resulted in specific erythroid progenitor cell differentiation defects that were highlighted by changes in gene expression and morphology, which could be rescued by injection of sparc mRNA. Furthermore, a comparison of blood phenotypes of sparc and fgfs knockdowns with similar defects and the sparc rescue of the fgf21 blood phenotype places sparc downstream of fgf21 in the genetic network regulating haematopoiesis in zebrafish. These results establish a role for an ECM protein (Sparc) as an important regulator of embryonic haematopoiesis during early development in zebrafish.

  5. Novel functions of the matricellular proteins osteopontin and osteonectin/SPARC.

    PubMed

    Sodek, Jaro; Zhu, Baoqian; Huynh, My-Hang; Brown, Theodore J; Ringuette, Maurice

    2002-01-01

    Osteopontin (OPN) and osteonectin/SPARC (ON/SPARC) are prominent matricellular components of the extracellular matrix of mineralized tissues of bones and teeth in which they can regulate the formation and growth of hydroxyapatite crystals and influence a variety of cell activities. OPN regulates cell responses through several integrin receptors and is also a ligand for the CD44 receptor, through which it acts as a chemoattractant. Although a cell-surface receptor for SPARC has not been identified it can block cell-cell and cell-matrix interactions and inhibit cell migration and chemotaxis. OPN and SPARC also appear to function inside cells. Thus, OPN appears to exist in association with the CD44 receptor inside migratory cells, while intracellular SPARC is associated with axonemal tubulin in ciliated epithelial cells. Analyses of fibroblasts and peritoneal macrophages from OPN-null and CD44-null cells show impaired functionality involving migration and cell fusion required for osteoclast formation, while disruption of SPARC expression leads to developmental defects in Xenopus. To gain further insights into the intracellular functions of OPN and SPARC, we have used the yeast two-hybrid system to identify potential interacting molecules. Using full-length SPARC as bait the carboxy-terminal domain, which contains two EF-hand, high-affinity binding sites, was found to have transcriptional activity, while several novel proteins that interact with the amino-terminal domains of SPARC and full-length OPN have been identified. The identification of OPN and SPARC inside specialized cells introduces a novel concept in cellular regulation by matricellular proteins.

  6. Endocytosis of Integrin-Binding Human Picornaviruses

    PubMed Central

    Merilahti, Pirjo; Koskinen, Satu; Heikkilä, Outi; Karelehto, Eveliina; Susi, Petri

    2012-01-01

    Picornaviruses that infect humans form one of the largest virus groups with almost three hundred virus types. They include significant enteroviral pathogens such as rhino-, polio-, echo-, and coxsackieviruses and human parechoviruses that cause wide range of disease symptoms. Despite the economic importance of picornaviruses, there are no antivirals. More than ten cellular receptors are known to participate in picornavirus infection, but experimental evidence of their role in cellular infection has been shown for only about twenty picornavirus types. Three enterovirus types and one parechovirus have experimentally been shown to bind and use integrin receptors in cellular infection. These include coxsackievirus A9 (CV-A9), echovirus 9, and human parechovirus 1 that are among the most common and epidemic human picornaviruses and bind to αV-integrins via RGD motif that resides on virus capsid. In contrast, echovirus 1 (E-1) has no RGD and uses integrin α2β1 as cellular receptor. Endocytosis of CV-A9 has recently been shown to occur via a novel Arf6- and dynamin-dependent pathways, while, contrary to collagen binding, E-1 binds inactive β1 integrin and enters via macropinocytosis. In this paper, we review what is known about receptors and endocytosis of integrin-binding human picornaviruses. PMID:23227048

  7. Endocytosis of integrin-binding human picornaviruses.

    PubMed

    Merilahti, Pirjo; Koskinen, Satu; Heikkilä, Outi; Karelehto, Eveliina; Susi, Petri

    2012-01-01

    Picornaviruses that infect humans form one of the largest virus groups with almost three hundred virus types. They include significant enteroviral pathogens such as rhino-, polio-, echo-, and coxsackieviruses and human parechoviruses that cause wide range of disease symptoms. Despite the economic importance of picornaviruses, there are no antivirals. More than ten cellular receptors are known to participate in picornavirus infection, but experimental evidence of their role in cellular infection has been shown for only about twenty picornavirus types. Three enterovirus types and one parechovirus have experimentally been shown to bind and use integrin receptors in cellular infection. These include coxsackievirus A9 (CV-A9), echovirus 9, and human parechovirus 1 that are among the most common and epidemic human picornaviruses and bind to αV-integrins via RGD motif that resides on virus capsid. In contrast, echovirus 1 (E-1) has no RGD and uses integrin α2β1 as cellular receptor. Endocytosis of CV-A9 has recently been shown to occur via a novel Arf6- and dynamin-dependent pathways, while, contrary to collagen binding, E-1 binds inactive β1 integrin and enters via macropinocytosis. In this paper, we review what is known about receptors and endocytosis of integrin-binding human picornaviruses.

  8. The matricellular protein CCN1 promotes mucosal healing in murine colitis through IL-6.

    PubMed

    Choi, J S; Kim, K-H; Lau, L F

    2015-11-01

    The matricellular protein CCN1 (CYR61) is known to function in wound healing and is upregulated in colons of patients with Crohn's disease and ulcerative colitis, yet its specific role in colitis is unknown. Here we have used Ccn1(dm/dm) knockin mice expressing a CCN1 mutant unable to bind integrins α6β1 and αMβ2 as a model to probe CCN1 function in dextran sodium sulfate (DSS)-induced colitis. Ccn1(dm/dm) mice exhibited high mortality, impaired mucosal healing, and diminished interleukin-6 (IL-6) expression during the repair phase of DSS-induced colitis compared with wild-type mice, despite having comparable severity of initial inflammation and tissue injury. CCN1-induced IL-6 expression in macrophages through integrin αMβ2 and in fibroblasts through α6β1, and IL-6 promoted intestinal epithelial cell (IEC) proliferation. Administration of purified CCN1 protein fully rescued Ccn1(dm/dm) mice from DSS-induced mortality, restored IEC proliferation and enhanced mucosal healing, whereas delivery of IL-6 partially rectified these defects. CCN1 therapy accelerated mucosal healing and recovery from DSS-induced colitis even in wild-type mice. These findings reveal a critical role for CCN1 in restoring mucosal homeostasis after intestinal injury in part through integrin-mediated induction of IL-6 expression, and suggest a therapeutic potential for activating the CCN1/IL-6 axis for treating inflammatory bowel disease.

  9. Expression of matricellular proteins in human uterine leiomyomas and normal myometrium.

    PubMed

    Bogusiewicz, Michal; Semczuk, Andrzej; Juszczak, Malgorzata; Langner, Ewa; Walczak, Katarzyna; Rzeski, Wojciech; Tomaszewski, Jacek; Rechberger, Tomasz

    2012-11-01

    Growth of human leiomyomas can probably be initiated as a response to injury, in a way similar to the development of keloids. Among many bioactive molecules, which are implicated in tissue repair, a pivotal role is attributed to matricellular proteins. The aim of the current study was to evaluate the immunohistochemical expression of tenascin-C (TNC), thrombospondin-1 (TSP-1), SPARC/osteonectin and tenascin-X (TNX) in human uterine leiomyomas and normal myometrium. Immunostaining was performed on 33 pairs of paraffin-fixed sections and 9 cell-lines derived from uterine leiomyomas and normal myometrium. Fifteen (45.5%) leiomyomas investigated were positive for TNC, whereas all normal myometrial samples were immunonegative (χ²=19.41; p<0.001). Immunostaining for TSP-1 was observed in 20 (60.6%) uterine fibroids and in 12 (36.4%) control samples (χ²=3.88; p<0.05). The expression of SPARC/osteonectin protein was more frequently found in leiomyomas than in normal myometrium, but this difference was not significant. Apart from one fibroid culture and one myometrial culture, all the others revealed strong TNC immunostaining. Expression of TSP-1 and SPARC/osteonectin was weak to moderate in all established cell-lines. None of the tissues or cell lines investigated showed positive staining for TNX. In conclusion, TSP-1 and TNC are likely to play important roles in the pathogenesis of uterine leiomyomas, presumably affecting cell proliferation and/or extracellular matrix deposition.

  10. The matricellular protein thrombospondin-1 globally regulates cardiovascular function and responses to stress via CD47.

    PubMed

    Roberts, David D; Miller, Thomas W; Rogers, Natasha M; Yao, Mingyi; Isenberg, Jeffrey S

    2012-04-01

    Matricellular proteins play diverse roles in modulating cell behavior by engaging specific cell surface receptors and interacting with extracellular matrix proteins, secreted enzymes, and growth factors. Studies of such interactions involving thrombospondin-1 have revealed several physiological functions and roles in the pathogenesis of injury responses and cancer, but the relatively mild phenotypes of mice lacking thrombospondin-1 suggested that thrombospondin-1 would not be a central player that could be exploited therapeutically. Recent research focusing on signaling through its receptor CD47, however, has uncovered more critical roles for thrombospondin-1 in acute regulation of cardiovascular dynamics, hemostasis, immunity, and mitochondrial homeostasis. Several of these functions are mediated by potent and redundant inhibition of the canonical nitric oxide pathway. Conversely, elevated tissue thrombospondin-1 levels in major chronic diseases of aging may account for the deficient nitric oxide signaling that characterizes these diseases, and experimental therapeutics targeting CD47 show promise for treating such chronic diseases as well as acute stress conditions that are associated with elevated thrombospondin-1 expression.

  11. Matricellular Protein Periostin Mediates Intestinal Inflammation through the Activation of Nuclear Factor κB Signaling.

    PubMed

    Koh, Seong-Joon; Choi, Younjeong; Kim, Byeong Gwan; Lee, Kook Lae; Kim, Dae Woo; Kim, Jung Ho; Kim, Ji Won; Kim, Joo Sung

    2016-01-01

    Periostin is a matricellular protein that interacts with various integrin molecules on the cell surface. Although periostin is expressed in inflamed colonic mucosa, its role in the regulation of intestinal inflammation remains unclear. We investigated the role of periostin in intestinal inflammation using Postn-deficient (Postn-/-) mice. Intestinal epithelial cells (IECs) were transfected by Postn small interfering RNAs. Periostin expression was determined in colon tissue samples from ulcerative colitis (UC) patients. Oral administration of dextran sulfate sodium (DSS) or rectal administration of trinitrobenzene sulfonic acid, induced severe colitis in wild-type mice, but not in Postn-/- mice. Administration of recombinant periostin induced colitis in Postn-/- mice. The periostin neutralizing-antibody ameliorated the severity of colitis in DSS-treated wild-type mice. Silencing of Postn inhibited inteleukin (IL)-8 mRNA expression and NF-κB DNA-binding activity in IECs. Tumor necrosis factor (TNF)-α upregulated mRNA expression of Postn in IECs, and recombinant periostin strongly enhanced IL-8 expression in combination with TNF-α, which was suppressed by an antibody against integrin αv (CD51). Periostin and CD51 were expressed at significantly higher levels in UC patients than in controls. Periostin mediates intestinal inflammation through the activation of NF-κB signaling, which suggests that periostin is a potential therapeutic target for inflammatory bowel disease.

  12. CyrA, a matricellular protein that modulates cell motility in Dictyostelium discoideum.

    PubMed

    Huber, Robert J; Suarez, Andres; O'Day, Danton H

    2012-05-01

    CyrA, an extracellular matrix (slime sheath), calmodulin (CaM)-binding protein in Dictyostelium discoideum, possesses four tandem EGF-like repeats in its C-terminus and is proteolytically cleaved during asexual development. A previous study reported the expression and localization of CyrA cleavage products CyrA-C45 and CyrA-C40. In this study, an N-terminal antibody was produced that detected the full-length 63kDa protein (CyrA-C63). Western blot analyses showed that the intracellular expression of CyrA-C63 peaked between 12 and 16h of development, consistent with the time that cells are developing into a motile, multicellular slug. CyrA immunolocalization and CyrA-GFP showed that the protein localized to the endoplasmic reticulum, particularly its perinuclear component. CyrA-C63 secretion began shortly after the onset of starvation peaking between 8 and 16h of development. A pharmacological analysis showed that CyrA-C63 secretion was dependent on intracellular Ca(2+) release and active CaM, PI3K, and PLA2. CyrA-C63 bound to CaM both intra- and extracellularly and both proteins were detected in the slime sheath deposited by migrating slugs. In keeping with its purported function, CyrA-GFP over-expression enhanced cAMP-mediated chemotaxis and CyrA-C45 was detected in vinculin B (VinB)-GFP immunoprecipitates, thus providing a link between the increase in chemotaxis and a specific cytoskeletal component. Finally, DdEGFL1-FITC was detected on the membranes of cells capped with concanavalin A suggesting that a receptor exists for this peptide sequence. Together with previous studies, the data presented here suggests that CyrA is a bona fide matricellular protein in D. discoideum.

  13. Degradome products of the matricellular protein CCN1 as modulators of pathological angiogenesis in the retina.

    PubMed

    Choi, Jinok; Lin, Ann; Shrier, Eric; Lau, Lester F; Grant, Maria B; Chaqour, Brahim

    2013-08-01

    CCN1 is a matricellular protein involved in normal vascular development and tissue repair. CCN1 exhibits cell- and context-dependent activities that are reflective of its tetramodular structure phylogenetically linked to four domains found in various matrix proteins. Here, we show that vitreal fluids from patients with proliferative diabetic retinopathy (PDR) were enriched with a two-module form of CCN1 comprising completely or partially the insulin-like growth factor-binding protein (IGFBP) and von Willebrand factor type C (vWC) domains. The two- and three-module forms comprising, in addition to IGFBP and vWC, the thrombospondin type 1 (TSP1) repeats are CCN1 degradome products by matrix metalloproteinase-2 and -14. The functional significance of CCN1 and its truncated variants was determined in the mouse model of oxygen-induced retinopathy, which simulates neovascular growth associated with PDR and assesses treatment outcomes. In this model, lentivirus-mediated expression of either CCN1 or the IGFBP-vWC-TSP1 form reduced ischemia-induced neovascularization, whereas ectopic expression of the IGFBP-vWC variant exacerbated pathological angiogenesis. The IGFBP-vWC form has potent proangiogenic properties promoting retinal endothelial cell growth, migration, and three-dimensional tubular structure formation, whereas the IGFBP-vWC-TSP1 variant suppressed cell growth and angiogenic gene expression. Both IGFBP-vWC and IGFBP-vWC-TSP1 forms exhibited predictable variations of their domain folding that enhanced their functional potential. These data provide new insights into the formation and activities of CCN1-truncated variants and raise the predictive value of the form containing completely or partially the IGFBP and vWC domains as a surrogate marker of CCN1 activity in PDR distinguishing pathological from physiological angiogenesis.

  14. The matricellular protein CCN1 suppresses hepatocarcinogenesis by inhibiting compensatory proliferation.

    PubMed

    Chen, C-C; Kim, K-H; Lau, L F

    2016-03-10

    Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related deaths worldwide, and is on the rise in the United States. Previous studies showed that the matricellular protein CCN1 (CYR61) is induced during hepatic injuries and functions to restrict and resolve liver fibrosis. Here, we show that CCN1 suppresses hepatocarcinogenesis by inhibiting carcinogen-induced compensatory hepatocyte proliferation, thus limiting the expansion of damaged and potentially oncogenic hepatocytes. Consistent with tumor suppression, CCN1 expression is downregulated in human HCC. Ccn1(ΔHep) mice with hepatocyte-specific deletion of Ccn1 suffer increased HCC tumor multiplicity induced by the hepatocarcinogen diethylnitrosamine (DEN). Knockin mice (Ccn1(dm/dm)) that express an integrin α6β1-binding defective CCN1 phenocopied Ccn1(ΔHep) mice, indicating that CCN1 acts through its α6β1 binding sites in this context. CCN1 effectively inhibits epidermal growth factor receptor (EGFR)-dependent hepatocyte proliferation through integrin α6-mediated accumulation of reactive oxygen species (ROS), thereby triggering p53 activation and cell cycle block. Consequently, Ccn1(dm/dm) mice exhibit diminished p53 activation and elevated compensatory hepatocyte proliferation, resulting in increased HCC. Furthermore, we show that a single dose of the EGFR inhibitor erlotinib delivered prior to DEN-induced injury was sufficient to block compensatory proliferation and annihilate development of HCC nodules observed 8 months later, suggesting potential chemoprevention by targeting CCN1-inhibitable EGFR-dependent hepatocyte proliferation. Together, these results show that CCN1 is an injury response protein that functions not only to restrict fibrosis in the liver, but also to suppress hepatocarcinogenesis by inhibiting EGFR-dependent hepatocyte compensatory proliferation.

  15. The matricellular protein CCN1 regulates TNF-α induced vascular endothelial cell apoptosis.

    PubMed

    Zhang, Jin; Wu, Gongxiong; Dai, Haibin

    2016-01-01

    Due to the epidemic obesity and associated diabetes, the incidence of atherosclerosis is increasing worldwide. Atherosclerosis is a chronic inflammatory disease characterized by the hardening and narrowing of arteries with plaques that consist of inflammatory cells, dead endothelial cells, lipids, and often hyper proliferated vascular smooth muscle cells. During the development of atherosclerosis, vascular endothelial cell (EC) apoptosis induced by the adipokine tumor necrosis factor alpha (TNF-α), is an early event in the plaque formation. However, TNF-α alone is not sufficient to induce apoptosis of endothelial cells. Recent studies suggested that the matricellular protein CCN family member 1 (CCN1) involves in endothelial cell dysfunction besides its well-known angiogenic function during tissue repair by promoting vascular smooth muscle cells proliferation and migration. Herein, we explored the possibility and mechanism of CCN1 in TNF-α induced endothelial cells apoptosis. Both mRNA and protein levels of CCN1 are found up-regulated in endothelial cells after TNF-α treatment. In addition, overexpression of CCN1 promoted endothelial cell apoptosis in the presence of TNF-α. Furthermore, CCN1 directly up-regulated the expression of TNF-α-target genes, and this up-regulation required the activation of P53 and NF-κB both in vivo and in vitro. Taken together, CNN1 regulates TNF-α induced endothelial cells apoptosis that may underlie poor response to TNF-α therapy and hence may be a better therapeutic target for preventing vascular dysfunction in obesity.

  16. Matricellular protein Cyr61 bridges lysophosphatidic acid and integrin pathways leading to cell migration.

    PubMed

    Wu, Daniel Dongwei; Zhang, Fuqiang; Hao, Feng; Chun, Jerold; Xu, Xuemin; Cui, Mei-Zhen

    2014-02-28

    Lysophosphatidic acid (LPA), a potent bioactive lipid found in atherosclerotic lesions, markedly induces smooth muscle cell (SMC) migration, which is an important process in atherogenesis. Therefore, understanding the mechanism of LPA-induced SMC migration is important. Several microarray databases suggest that the matricellular protein Cyr61 is highly induced by LPA. We hypothesized that Cyr61 mediates LPA-induced cell migration. Our data show that LPA induced temporal and spatial expression of Cyr61, which promptly accumulated in the cellular Golgi apparatus and then translocated to the extracellular matrix. Cyr61 antibody blockade and siRNA inhibition both diminished LPA-induced SMC migration, indicating a novel regulatory role of Cyr61. SMCs derived from LPA receptor 1 (LPA1) knock-out mice lack the ability of Cyr61 induction and cell migration, supporting the concept that LPA1 is required for Cyr61 expression and migration. By contrast, PPARγ was not found to be involved in LPA-mediated effects. Furthermore, focal adhesion kinase (FAK), a nonreceptor tyrosine kinase important for regulating cell migration, was activated by LPA at a late time frame coinciding with Cyr61 accumulation. Interestingly, knockdown of Cyr61 blocked LPA-induced FAK activation, indicating that an LPA-Cyr61-FAK axis leads to SMC migration. Our results further demonstrate that plasma membrane integrins α6β1 and ανβ3 transduced the LPA-Cyr61 signal toward FAK activation and migration. Taken together, these data reveal that de novo Cyr61 in the extracellular matrix bridges LPA and integrin pathways, which in turn, activate FAK, leading to cell migration. The current study provides new insights into mechanisms underlying cell migration-related disorders, including atherosclerosis, restenosis, and cancers.

  17. Utilizing Fibronectin Integrin-Binding Specificity to Control Cellular Responses

    PubMed Central

    Bachman, Haylee; Nicosia, John; Dysart, Marilyn; Barker, Thomas H.

    2015-01-01

    Significance: Cells communicate with the extracellular matrix (ECM) protein fibronectin (Fn) through integrin receptors on the cell surface. Controlling integrin–Fn interactions offers a promising approach to directing cell behavior, such as adhesion, migration, and differentiation, as well as coordinated tissue behaviors such as morphogenesis and wound healing. Recent Advances: Several different groups have developed recombinant fragments of Fn that can control epithelial to mesenchymal transition, sequester growth factors, and promote bone and wound healing. It is thought that these physiological responses are, in part, due to specific integrin engagement. Furthermore, it has been postulated that the integrin-binding domain of Fn is a mechanically sensitive switch that drives binding of one integrin heterodimer over another. Critical Issues: Although computational simulations have predicted the mechano-switch hypothesis and recent evidence supports the existence of varying strain states of Fn in vivo, experimental evidence of the Fn integrin switch is still lacking. Future Directions: Evidence of the integrin mechano-switch will enable the development of new Fn-based peptides in tissue engineering and wound healing, as well as deepen our understanding of ECM pathologies, such as fibrosis. PMID:26244106

  18. The matricellular protein CYR61 interferes with normal pancreatic islets architecture and promotes pancreatic neuroendocrine tumor progression.

    PubMed

    Huang, Yu-Ting; Lan, Qiang; Ponsonnet, Lionel; Blanquet, Marisa; Christofori, Gerhard; Zaric, Jelena; Rüegg, Curzio

    2016-01-12

    The significance of matricellular proteins during development and cancer progression is widely recognized. However, how these proteins actively contribute to physiological development and pathological cancer progression is only partially elucidated. In this study, we investigated the role of the matricellular protein Cysteine-rich 61 (CYR61) in pancreatic islet development and carcinogenesis. Transgenic expression of CYR61 in β cells (Rip1CYR mice) caused irregular islets morphology and distorted sorting of α cells, but did not alter islets size, number or vascularization. To investigate the function of CYR61 during carcinogenesis, we crossed Rip1CYR mice with Rip1Tag2 mice, a well-established model of β cell carcinogenesis. Beta tumors in Rip1Tag2CYR mice were larger, more invasive and more vascularized compared to tumors in Rip1Tag2 mice. The effect of CYR61 on angiogenesis was fully abrogated by treating mice with the anti-VEGFR2 mAb DC101. Results from in vitro assays demonstrated that CYR61 modulated integrin α6β1-dependent invasion and adhesion without altering its expression. Taken together, these results show that CYR61 expression in pancreatic β cells interferes with normal islet architecture, promotes islet tumor growth, invasion and VEGF/VERGFR-2-dependent tumor angiogenesis. Taken together, these observations demonstrate that CYR61 acts as a tumor-promoting gene in pancreatic neuroendocrine tumors.

  19. Canine malignant melanoma alpha-3 integrin binding peptides

    PubMed Central

    Aina, Olulanu H.; Maeda, Yoshiko; Harrison, Matthew; Zwingenberger, Allison L.; Walker, Naomi J.; Lam, Kit S.; Kent, Michael S.

    2014-01-01

    There is a need to develop novel targeted imaging and therapeutic agents that can aid in early diagnosis, detection of metastasis and treatment of melanoma. Alpha-3 integrin is overexpressed in 82% of metastatic melanomas in humans and may be a potential target for peptide ligands carrying therapeutic agents. Five melanoma cell lines were generated from canine primary oral and metastatic canine tumors, grown in mice, and validated with melanoma markers Melan A, S-100, Micropthalmia transcription factor (MITF), Tyrosinase, and MART-1. The melanoma cell lines were tested for binding affinity to previously published alpha-3 integrin-binding peptides containing the cdGXGXXc motif. Fluorescent conjugates of the alpha-3 integrin binding OA02 peptide were used to quantify receptor affinity in the cell lines, a specimen of canine primary oral melanoma, and melanoma xenografts. Alpha-3 integrin was expressed by all 5 canine melanoma cell lines. Four of the 5 lines as well as the primary canine tumor showed affinity to alpha-3 integrin binding peptides with the cdGXGXXc motif. Optical imaging of canine melanoma xenografts in nude mice indicates rapid, strong uptake of the optical tracer in the tumor with an average persistence of approximately 48 hours. Ex vivo images showed high tumor-to-background ratio, with tumor signals more than twice that of the kidney and other vital organs. We propose that integrin alpha-3 integrin binding ligands could potentially become useful probes for imaging and delivery of cytotoxic agents for the treatment of melanoma. PMID:21722969

  20. Thymic epithelial cell expansion through matricellular protein CYR61 boosts progenitor homing and T-cell output.

    PubMed

    Emre, Yalin; Irla, Magali; Dunand-Sauthier, Isabelle; Ballet, Romain; Meguenani, Mehdi; Jemelin, Stephane; Vesin, Christian; Reith, Walter; Imhof, Beat A

    2013-01-01

    Thymic epithelial cells (TEC) are heterogeneous stromal cells that generate microenvironments required for the formation of T cells within the thymus. Defects in TEC lead to immunodeficiency or autoimmunity. Here we identify TEC as the major source of cysteine-rich protein 61 (CYR61), a matricellular protein implicated in cell proliferation and migration. Binding of CYR61 to LFA-1, ICAM-1 and integrin α6 supports the adhesion of TEC and thymocytes as well as their interaction. Treatment of thymic lobes with recombinant CYR61 expands the stromal compartment by inducing the proliferation of TEC and activates Akt signalling. Engraftment of CYR61-overexpressing thymic lobes into athymic nude mice drastically boosts the yield of thymic output via expansion of TEC. This increases the space for the recruitment of circulating hematopoietic progenitors and the development of T cells. Our discovery paves the way for therapeutic interventions designed to restore thymus stroma and T-cell generation.

  1. Thymic epithelial cell expansion through matricellular protein CYR61 boosts progenitor homing and T-cell output

    NASA Astrophysics Data System (ADS)

    Emre, Yalin; Irla, Magali; Dunand-Sauthier, Isabelle; Ballet, Romain; Meguenani, Mehdi; Jemelin, Stephane; Vesin, Christian; Reith, Walter; Imhof, Beat A.

    2013-11-01

    Thymic epithelial cells (TEC) are heterogeneous stromal cells that generate microenvironments required for the formation of T cells within the thymus. Defects in TEC lead to immunodeficiency or autoimmunity. Here we identify TEC as the major source of cysteine-rich protein 61 (CYR61), a matricellular protein implicated in cell proliferation and migration. Binding of CYR61 to LFA-1, ICAM-1 and integrin α6 supports the adhesion of TEC and thymocytes as well as their interaction. Treatment of thymic lobes with recombinant CYR61 expands the stromal compartment by inducing the proliferation of TEC and activates Akt signalling. Engraftment of CYR61-overexpressing thymic lobes into athymic nude mice drastically boosts the yield of thymic output via expansion of TEC. This increases the space for the recruitment of circulating hematopoietic progenitors and the development of T cells. Our discovery paves the way for therapeutic interventions designed to restore thymus stroma and T-cell generation.

  2. The role of the matricellular protein SPARC in the dynamic interaction between the tumor and the host.

    PubMed

    Podhajcer, Osvaldo L; Benedetti, Lorena; Girotti, Maria Romina; Prada, Federico; Salvatierra, Edgardo; Llera, Andrea S

    2008-09-01

    Tumor growth is essentially the result of an evolving cross-talk between malignant and surrounding stromal cells (fibroblasts, endothelial cells and inflammatory cells). This heterogeneous mass of extracellular matrix and intermingled cells interact through cell-cell and cell-matrix contacts. Malignant cells also secrete soluble proteins that reach neighbor stromal cells, forcing them to provide the soil on which they will grow and metastasize. Different studies including expression array analysis identified the matricellular protein SPARC as a marker of poor prognosis in different cancer types. Further evidence demonstrated that high SPARC levels are often associated with the most aggressive and highly metastatic tumors. Here we describe the most recent evidence that links SPARC with human cancer progression, the controversy regarding its role in certain human cancers and the physiological processes in which SPARC is involved: epithelial-mesenchymal transition, immune surveillance and angiogenesis. Its relevance as a potential target in cancer therapy is also discussed.

  3. Stromal niche communalities underscore the contribution of the matricellular protein SPARC to B-cell development and lymphoid malignancies.

    PubMed

    Sangaletti, Sabina; Tripodo, Claudio; Portararo, Paola; Dugo, Matteo; Vitali, Caterina; Botti, Laura; Guarnotta, Carla; Cappetti, Barbara; Gulino, Alessandro; Torselli, Ilaria; Casalini, Patrizia; Chiodoni, Claudia; Colombo, Mario P

    2014-01-01

    Neoplastic B-cell clones commonly arise within secondary lymphoid organs (SLO). However, during disease progression, lymphomatous cells may also colonize the bone marrow (BM), where they localize within specialized stromal niches, namely the osteoblastic and the vascular niche, according to their germinal center- or extra-follicular-derivation, respectively. We hypothesized the existence of common stromal motifs in BM and SLO B-cell lymphoid niches involved in licensing normal B-cell development as well as in fostering transformed B lymphoid cells. Thus, we tested the expression of prototypical mesenchymal stromal cell (MSC) markers and regulatory matricellular proteins in human BM and SLO under physiologically unperturbed conditions and during B-cell lymphoma occurrence. We identified common stromal features in the BM osteoblastic niche and SLO germinal center (GC) microenvironments, traits that were also enriched within BM infiltrates of GC-associated B-cell lymphomas, suggesting that stromal programs involved in central and peripheral B-cell lymphopoiesis are also involved in malignant B-cell nurturing. Among factors co-expressed by stromal elements within these different specialized niches, we identified the pleiotropic matricellular protein secreted protein acidic and rich in cysteine (SPARC). The actual role of stromal SPARC in normal B-cell lymphopoiesis, investigated in Sparc(-/-) mice and BM chimeras retaining the Sparc(-/-) genotype in host stroma, demonstrated defective BM and splenic B-cell lymphopoiesis. Moreover, in the Trp53 knockout (KO) lymphoma model, p53(-/-)/Sparc(-/-) double-KO mice displayed impaired spontaneous splenic B-cell lymphomagenesis and reduced neoplastic clone BM infiltration in comparison with their p53(-/-)/Sparc(+/+) counterparts. Our results are among the first to demonstrate the existence of common stromal programs regulating both the BM osteoblastic niche and the SLO GC lymphopoietic functions potentially fostering the genesis

  4. Stromal niche communalities underscore the contribution of the matricellular protein SPARC to B-cell development and lymphoid malignancies.

    PubMed

    Sangaletti, Sabina; Tripodo, Claudio; Portararo, Paola; Dugo, Matteo; Vitali, Caterina; Botti, Laura; Guarnotta, Carla; Cappetti, Barbara; Gulino, Alessandro; Torselli, Ilaria; Casalini, Patrizia; Chiodoni, Claudia; Colombo, Mario P

    2014-01-01

    Neoplastic B-cell clones commonly arise within secondary lymphoid organs (SLO). However, during disease progression, lymphomatous cells may also colonize the bone marrow (BM), where they localize within specialized stromal niches, namely the osteoblastic and the vascular niche, according to their germinal center- or extra-follicular-derivation, respectively. We hypothesized the existence of common stromal motifs in BM and SLO B-cell lymphoid niches involved in licensing normal B-cell development as well as in fostering transformed B lymphoid cells. Thus, we tested the expression of prototypical mesenchymal stromal cell (MSC) markers and regulatory matricellular proteins in human BM and SLO under physiologically unperturbed conditions and during B-cell lymphoma occurrence. We identified common stromal features in the BM osteoblastic niche and SLO germinal center (GC) microenvironments, traits that were also enriched within BM infiltrates of GC-associated B-cell lymphomas, suggesting that stromal programs involved in central and peripheral B-cell lymphopoiesis are also involved in malignant B-cell nurturing. Among factors co-expressed by stromal elements within these different specialized niches, we identified the pleiotropic matricellular protein secreted protein acidic and rich in cysteine (SPARC). The actual role of stromal SPARC in normal B-cell lymphopoiesis, investigated in Sparc(-/-) mice and BM chimeras retaining the Sparc(-/-) genotype in host stroma, demonstrated defective BM and splenic B-cell lymphopoiesis. Moreover, in the Trp53 knockout (KO) lymphoma model, p53(-/-)/Sparc(-/-) double-KO mice displayed impaired spontaneous splenic B-cell lymphomagenesis and reduced neoplastic clone BM infiltration in comparison with their p53(-/-)/Sparc(+/+) counterparts. Our results are among the first to demonstrate the existence of common stromal programs regulating both the BM osteoblastic niche and the SLO GC lymphopoietic functions potentially fostering the genesis

  5. Identification of a sequence in the matricellular protein SPARC that interacts with the scavenger receptor stabilin-1.

    PubMed

    Workman, Gail; Sage, E Helene

    2011-04-01

    SPARC (osteonectin/BM-40), a secreted matricellular protein that promotes cellular deadhesion and motility in wound healing, carcinogenesis, and inflammation, binds to the scavenger receptor stabilin-1 in alternatively activated macrophages and undergoes endocytosis and clearance from the extracellular space. Both SPARC and stabilin-1 are expressed by endothelial cells during inflammation, but their interaction in this context is unknown. We have identified a binding site on SPARC for stabilin-1 by a solid-state peptide array coupled with a modified enzyme-linked immunosorbent assay. A monoclonal antibody that recognizes the identified binding site was also characterized that could be an inhibitor for the SPARC-stabilin-1 interaction in macrophages or endothelial cells.

  6. The matricellular protein CCN1 enhances TGF-β1/SMAD3-dependent profibrotic signaling in fibroblasts and contributes to fibrogenic responses to lung injury.

    PubMed

    Kurundkar, Ashish R; Kurundkar, Deepali; Rangarajan, Sunad; Locy, Morgan L; Zhou, Yong; Liu, Rui-Ming; Zmijewski, Jaroslaw; Thannickal, Victor J

    2016-06-01

    Matricellular proteins mediate pleiotropic effects during tissue injury and repair. CCN1 is a matricellular protein that has been implicated in angiogenesis, inflammation, and wound repair. In this study, we identified CCN1 as a gene that is differentially up-regulated in alveolar mesenchymal cells of human subjects with rapidly progressive idiopathic pulmonary fibrosis (IPF). Elevated levels of CCN1 mRNA were confirmed in lung tissues of IPF subjects undergoing lung transplantation, and CCN1 protein was predominantly localized to fibroblastic foci. CCN1 expression in ex vivo IPF lung fibroblasts correlated with gene expression of the extracellular matrix proteins, collagen (Col)1a1, Col1a2, and fibronectin as well as the myofibroblast marker, α-smooth muscle actin. RNA interference (RNAi)-mediated knockdown of CCN1 down-regulated the constitutive expression of these profibrotic genes in IPF fibroblasts. TGF-β1, a known mediator of tissue fibrogenesis, induces gene and protein expression of CCN1 via a mothers against decapentaplegic homolog 3 (SMAD3)-dependent mechanism. Importantly, endogenous CCN1 potentiates TGF-β1-induced SMAD3 activation and induction of profibrotic genes, supporting a positive feedback loop leading to myofibroblast activation. In vivo RNAi-mediated silencing of CCN1 attenuates fibrogenic responses to bleomycin-induced lung injury. These studies support previously unrecognized, cooperative interaction between the CCN1 matricellular protein and canonical TGF-β1/SMAD3 signaling that promotes lung fibrosis.-Kurundkar, A. R., Kurundkar, D., Rangarajan, S., Locy, M. L., Zhou, Y., Liu, R.-M., Zmijewski, J., Thannickal, V. J. The matricellular protein CCN1 enhances TGF-β1/SMAD3-dependent profibrotic signaling in fibroblasts and contributes to fibrogenic responses to lung injury.

  7. Integrin binding by B orrelia burgdorferi  P66 facilitates dissemination but is not required for infectivity

    PubMed Central

    Ristow, Laura C.; Bonde, Mari; Lin, Yi‐Pin; Sato, Hiromi; Curtis, Michael; Wesley, Erin; Hahn, Beth L.; Fang, Juan; Wilcox, David A.; Leong, John M.; Bergström, Sven

    2015-01-01

    Summary P66, a B orrelia burgdorferi surface protein with porin and integrin‐binding activities, is essential for murine infection. The role of P66 integrin‐binding activity in B . burgdorferi infection was investigated and found to affect transendothelial migration. The role of integrin binding, specifically, was tested by mutation of two amino acids (D205A,D207A) or deletion of seven amino acids (Del202–208). Neither change affected surface localization or channel‐forming activity of P66, but both significantly reduced binding to αvβ3. Integrin‐binding deficient B . burgdorferi strains caused disseminated infection in mice at 4 weeks post‐subcutaneous inoculation, but bacterial burdens were significantly reduced in some tissues. Following intravenous inoculation, the Del202–208 bacteria were below the limit of detection in all tissues assessed at 2 weeks post‐inoculation, but bacterial burdens recovered to wild‐type levels at 4 weeks post‐inoculation. The delay in tissue colonization correlated with reduced migration of the Del202–208 strains across microvascular endothelial cells, similar to Δp66 bacteria. These results indicate that integrin binding by P66 is important to efficient dissemination of B . burgdorferi, which is critical to its ability to cause disease manifestations in incidental hosts and to its maintenance in the enzootic cycle. PMID:25604835

  8. The influence of surface integrin binding patterns on specific biomaterial-cell interactions

    NASA Astrophysics Data System (ADS)

    Beranek, Maggi Marie

    As the future of biomaterials progresses toward bioactivity, the biomaterial surface must control non-specific protein adsorption and encourage selective protein and cell adsorption. Integrins alphavbeta3, alpha 1beta1, alpha5beta1 and alpha Mbeta2 are expressed on cells involved in endothelialization, inflammation, and intimal hyperplasia. These cellular events play a vital role in biomaterial biocompatibility, especially in the vascular environment. The overall hypothesis of these studies is that biomaterial surfaces exhibit selective integrin binding, which then specifies differential cell binding. To test this hypothesis, four specific aims were developed. The first aim was designed to determine whether metal and polymeric biomaterials exhibit selective integrin binding. The tested materials included 316L stainless steel, nitinol, gold, Elgiloy RTM, poly(D, L-lactide-co-glycolide), polycarbonate urethane and expanded polytetrafluoroethylene. Discrete integrin binding patterns were detected microscopically using integrin specific fluorescent antibodies. Stainless steel exhibited high level integrin alpha1beta 1 and low level integrin alphaMbeta2 binding pattern. This suggests that this metal surface should selectively encourage endothelial cell to inflammatory cell binding. In contrast, gold bound ten times the amount of integrin alphaMbeta2 compared to integrin alpha1beta1, which should encourage inflammatory cell adhesion. The 65/35 poly(D, L-lactide-co-glycolide) was the only polymeric biomaterial tested that had integrin binding levels comparable to metal biomaterials. Based on these observations, a combinational biomaterial with a surface pattern of 65/35 poly(D, L-lactide-co-glycolide) dots on a 316L stainless steel background was created. A pattern of high level integrin alpha1beta1 binding and low level integrin alpha Mbeta2 binding on this combinational surface indicates that this surface should selectively favor endothelial cell binding. In the second

  9. The Matricellular Protein CCN1/CYR61 Induces Fibroblast Senescence and Restricts Fibrosis in Cutaneous Wound Healing

    PubMed Central

    Jun, Joon-Il; Lau, Lester F.

    2010-01-01

    Cellular senescence is a recognised mechanism of tumor suppression; however, its contribution to other pathologies is not well understood. We show that the matricellular protein CCN1/CYR61, which is dynamically expressed at sites of wound repair, can induce fibroblast senescence through its cell adhesion receptors, integrin α6β1 and heparan sulfate proteoglycans. CCN1 induces DNA damage response and p53 activation, and activates the RAC1-NOX1 complex to induce reactive oxygen species (ROS) generation and ROS-dependent activation of the p16INK4a/pRb pathway, leading to senescence and concomitant expression of antifibrotic genes. Senescent fibroblasts accumulate in granulation tissues of healing cutaneous wounds and express antifibrotic genes in wild type mice. These processes are obliterated in knockin mice that express a senescence-defective CCN1 mutant, resulting in exacerbated fibrosis. Topical application of CCN1 protein to wounds reverses these defects. Thus, fibroblast senescence is a CCN1-dependent wound healing response in cutaneous injury, functioning to curb fibrosis during tissue repair. PMID:20526329

  10. Matricellular protein CCN1 promotes regression of liver fibrosis through induction of cellular senescence in hepatic myofibroblasts.

    PubMed

    Kim, Ki-Hyun; Chen, Chih-Chiun; Monzon, Ricardo I; Lau, Lester F

    2013-05-01

    Liver fibrosis occurs as a wound-healing response to chronic hepatic injuries irrespective of the underlying etiology and may progress to life-threatening cirrhosis. Here we show that CCN1, a matricellular protein of the CCN (CYR61/CTGF/NOV) family, is accumulated in hepatocytes of human cirrhotic livers. CCN1 is not required for liver development or regeneration, since these processes are normal in mice with hepatocyte-specific Ccn1 deletion. However, Ccn1 expression is upregulated upon liver injuries and functions to inhibit liver fibrogenesis induced by either carbon tetrachloride intoxication or bile duct ligation and promote fibrosis regression. CCN1 acts by triggering cellular senescence in activated hepatic stellate cells and portal fibroblasts by engaging integrin α6β1 to induce reactive oxygen species accumulation through the RAC1-NADPH oxidase 1 enzyme complex, whereupon the senescent cells express an antifibrosis genetic program. Mice with hepatocyte-specific Ccn1 deletion suffer exacerbated fibrosis with a concomitant deficit in cellular senescence, whereas overexpression of hepatic Ccn1 reduces liver fibrosis with enhanced senescence. Furthermore, tail vein delivery of purified CCN1 protein accelerates fibrosis regression in mice with established fibrosis. These findings reveal a novel integrin-dependent mechanism of fibrosis resolution in chronic liver injury and identify the CCN1 signaling pathway as a potential target for therapeutic intervention.

  11. The matricellular protein Cyr61 is a key mediator of platelet-derived growth factor-induced cell migration.

    PubMed

    Zhang, Fuqiang; Hao, Feng; An, Dong; Zeng, Linlin; Wang, Yi; Xu, Xuemin; Cui, Mei-Zhen

    2015-03-27

    Platelet-derived growth factor (PDGF), a potent chemoattractant, induces cell migration via the MAPK and PI3K/Akt pathways. However, the downstream mediators are still elusive. In particular, the role of extracellular mediators is largely unknown. In this study, we identified the matricellular protein Cyr61, which is de novo synthesized in response to PDGF stimulation, as the key downstream mediator of the ERK and JNK pathways, independent of the p38 MAPK and AKT pathways, and, thereby, it mediates PDGF-induced smooth muscle cell migration but not proliferation. Our results revealed that, when Cyr61 was newly synthesized by PDGF, it was promptly translocated to the extracellular matrix and physically interacted with the plasma membrane integrins α6β1 and αvβ3. We further demonstrate that Cyr61 and integrins are integral components of the PDGF signaling pathway via an "outside-in" signaling route to activate intracellular focal adhesion kinase (FAK), leading to cell migration. Therefore, this study provides the first evidence that the PDGF-induced endogenous extracellular matrix component Cyr61 is a key mediator in modulating cell migration by connecting intracellular PDGF-ERK and JNK signals with integrin/FAK signaling. Therefore, extracellular Cyr61 convergence with growth factor signaling and integrin/FAK signaling is a new concept of growth factor-induced cell migration. The discovered signaling pathway may represent an important therapeutic target in growth factor-mediated cell migration/invasion-related vascular diseases and tumorigenesis.

  12. Regulation of beta catenin signaling and parathyroid hormone anabolic effects in bone by the matricellular protein periostin.

    PubMed

    Bonnet, Nicolas; Conway, Simon J; Ferrari, Serge L

    2012-09-11

    Periostin (Postn) is a matricellular protein preferentially expressed by osteocytes and periosteal osteoblasts in response to mechanical stimulation and parathyroid hormone (PTH). Whether and how periostin expression influences bone anabolism, however, remains unknown. We investigated the skeletal response of adult Postn(-/-) and Postn(+/+) mice to intermittent PTH. Compared with Postn(+/+), Postn(-/-) mice had a lower bone mass, cortical bone volume, and strength response to PTH. PTH-stimulated bone-forming indices were all significantly lower in Postn(-/-) mice, particularly at the periosteum. Furthermore, in vivo stimulation of Wnt-β-catenin signaling by PTH, as evaluated in TOPGAL reporter mice, was inhibited in the absence of periostin (TOPGAL;Postn(-/-) mice). PTH stimulated periostin and inhibited MEF2C and sclerostin (Sost) expression in bone and osteoblasts in vitro. Recombinant periostin also suppressed Sost expression, which was mediated through the integrin αVβ3 receptor, whereas periostin-blocking antibody prevented inhibition of MEF2C and Sost by PTH. In turn, administration of a Sost-blocking antiboby partially restored the PTH-mediated increase in bone mass in Postn(-/-) mice. In addition, primary osteoblasts from Postn(-/-) mice showed a lower proliferation, mineralization, and migration, both spontaneously and in response to PTH. Osteoblastic gene expression levels confirmed a defect of Postn(-/-) osteoblast differentiation with and without PTH, as well as an increased osteoblast apoptosis in the absence of periostin. These data elucidate the complex role of periostin on bone anabolism, through the regulation of Sost, Wnt-β-catenin signaling, and osteoblast differentiation.

  13. Matricellular protein CCN1 (CYR61) expression is associated with high-grade ductal carcinoma in situ.

    PubMed

    Saglam, Ozlen; Dai, Feng; Husain, Seema; Zhan, Yilei; Toruner, Gokce; Haines, G Kenneth

    2014-06-01

    Cysteine-rich protein 61, connective tissue growth factor, and nephroblastoma overexpressed gene (CCN) comprise a family of matricellular proteins that have multiple physiologic functions including development, tissue repair, cell adhesion, migration, and proliferation. The expression of CCN1, cyclin D1, β-catenin, and p53 was explored by immunohistochemistry in different grades of ductal carcinoma in situ (DCIS) cases. These cases did not contain any infiltrating carcinoma components. In addition, all cysteine-rich protein 61 gene exons (encoding the CCN1 protein) were sequenced in 30 samples. Allred and H-scores were calculated for expression in both DCIS and the surrounding benign breast tissue. All cases of DCIS showed degrees of cytoplasmic CCN1 staining with median H-scores of 170, 160, and 60 in grades 3, 2, and 1, respectively (P = .043). Twelve of 28 DCIS 3, 1 of 15 DCIS 2, and 0 of 18 DCIS 1 also showed nuclear staining for CCN1. The cytoplasmic staining difference was preserved when the cases were divided into estrogen receptor (ER)+/DCIS grade 1, ER+/DCIS 2 and 3, and ER-/DCIS 2 and 3 by the H-score (P = .037). Cyclin D1 expression was positively correlated with the CCN1 cytoplasmic H-score in all DCIS samples (P = .038). Membranous β-catenin expression correlated with the grade of intraepithelial carcinoma by both H-score (P = .047) and Allred score (P = .026). Our results suggest that CCN1 has a role in the development of intraepithelial carcinoma. CCN1 expression correlates with grade of DCIS independent of ER status. It can induce cell cycle progression through cyclin D1. It is warranted to study high expression of CCN1 in DCIS as an independent risk factor in a larger cohort.

  14. The matricellular protein CYR61 inhibits osteoclastogenesis by a mechanism independent of alphavbeta3 and alphavbeta5.

    PubMed

    Crockett, Julie C; Schütze, Norbert; Tosh, Denise; Jatzke, Susanne; Duthie, Angela; Jakob, Franz; Rogers, Michael J

    2007-12-01

    Cysteine-rich protein 61 (CYR61/CCN1) belongs to the family of CCN matricellular proteins. Most of the known effects of CCN proteins appear to be due to binding to extracellular growth factors or integrins, including alpha(v)beta(3) and alpha(v)beta(5). Although CYR61 can stimulate osteoblast differentiation, until now the effect of CYR61 on osteoclasts was unknown. We demonstrate that recombinant human CYR61 inhibits the formation of multinucleated, alpha(v)beta(3)-positive, or tartrate-resistant acid phosphatase-positive human, mouse, and rabbit osteoclasts in vitro. CYR61 markedly reduced the expression of the osteoclast phenotypic markers tartrate-resistant acid phosphatase, matrix metalloproteinase-9, calcitonin receptor, and cathepsin K. However, CYR61 did not affect the formation of multinucleated osteoclasts when added to osteoclast precursors prior to fusion or affect the number or resorptive activity of osteoclasts cultured on dentine discs, indicating that CYR61 affects early osteoclast precursors but not mature osteoclasts. CYR61 did not affect receptor activator of nuclear factor-kappaB (RANK) ligand-induced phosphorylation of p38 or ERK1/2 in human macrophages and did not affect RANK ligand-induced activation of nuclear factor-kappaB, indicating that CYR61 does not appear to inhibit osteoclastogenesis by affecting RANK signaling. Furthermore, a mutant form of CYR61 defective in binding to alpha(v)beta(3) also inhibited osteoclastogenesis, and CYR61 inhibited osteoclastogenesis similarly in cultures of mouse wild-type or beta(5)(-/-) macrophages. Thus, CYR61 does not appear to inhibit osteoclast formation by interacting with alpha(v)beta(3) or alpha(v)beta(5). These observations demonstrate that CYR61 is a hitherto unrecognized inhibitor of osteoclast formation, although the exact mechanism of inhibition remains to be determined. Given that CYR61 also stimulates osteoblasts, CYR61 could represent an important bifunctional local regulator of bone

  15. The matricellular protein CCN1/Cyr61 is a critical regulator of Sonic Hedgehog in pancreatic carcinogenesis.

    PubMed

    Haque, Inamul; De, Archana; Majumder, Monami; Mehta, Smita; McGregor, Douglas; Banerjee, Sushanta K; Van Veldhuizen, Peter; Banerjee, Snigdha

    2012-11-01

    CCN1 is a matricellular protein and a member of the CCN family of growth factors. CCN1 is associated with the development of various cancers including pancreatic ductal adenocarcinoma (PDAC). Our recent studies found that CCN1 plays a critical role in pancreatic carcinogenesis through the induction of EMT and stemness. CCN1 mRNA and protein were detected in the early precursor lesions, and their expression intensified with disease progression. However, biochemical activity and the molecular targets of CCN1 in pancreatic cancer cells are unknown. Here we show that CCN1 regulates the Sonic Hedgehog (SHh) signaling pathway, which is associated with the PDAC progression and poor prognosis. SHh regulation by CCN1 in pancreatic cancer cells is mediated through the active Notch-1. Notably, active Notch-1is recruited by CCN1 in these cells via the inhibition of proteasomal degradation results in stabilization of the receptor. We find that CCN1-induced activation of SHh signaling might be necessary for CCN1-dependent in vitro pancreatic cancer cell migration and tumorigenicity of the side population of pancreatic cancer cells (cancer stem cells) in a xenograft in nude mice. Moreover, the functional role of CCN1 could be mediated through the interaction with the αvβ3 integrin receptor. These extensive studies propose that targeting CCN1 can provide a new treatment option for patients with pancreatic cancer since blocking CCN1 simultaneously blocks two critical pathways (i.e. SHh and Notch1) associated with the development of the disease as well as drug resistance.

  16. Label-free mass spectrometric analysis of the mdx-4cv diaphragm identifies the matricellular protein periostin as a potential factor involved in dystrophinopathy-related fibrosis.

    PubMed

    Holland, Ashling; Dowling, Paul; Meleady, Paula; Henry, Michael; Zweyer, Margit; Mundegar, Rustam R; Swandulla, Dieter; Ohlendieck, Kay

    2015-07-01

    Proteomic profiling plays a decisive role in the identification of novel biomarkers of muscular dystrophy and the elucidation of new pathobiochemical mechanisms that underlie progressive muscle wasting. Building on the findings of recent comparative analyses of tissue samples and body fluids from dystrophic animals and patients afflicted with Duchenne muscular dystrophy, we have used here label-free MS to study the severely dystrophic diaphragm from the not extensively characterized mdx-4cv mouse. This animal model of progressive muscle wasting exhibits less dystrophin-positive revertant fibers than the conventional mdx mouse, making it ideal for the future monitoring of experimental therapies. The pathoproteomic signature of the mdx-4cv diaphragm included a significant increase in the fibrosis marker collagen and related extracellular matrix proteins (asporin, decorin, dermatopontin, prolargin) and cytoskeletal proteins (desmin, filamin, obscurin, plectin, spectrin, tubulin, vimentin, vinculin), as well as decreases in proteins of ion homeostasis (parvalbumin) and the contractile apparatus (myosin-binding protein). Importantly, one of the most substantially increased proteins was identified as periostin, a matricellular component and apparent marker of fibrosis and tissue damage. Immunoblotting confirmed a considerable increase of periostin in the dystrophin-deficient diaphragm from both mdx and mdx-4cv mice, suggesting an involvement of this matricellular protein in dystrophinopathy-related fibrosis.

  17. Treatment with the matricellular protein CCN3 blocks and/or reverses fibrosis development in obesity with diabetic nephropathy.

    PubMed

    Riser, Bruce L; Najmabadi, Feridoon; Garchow, Kendra; Barnes, Jeffrey L; Peterson, Darryl R; Sukowski, Ernest J

    2014-11-01

    Fibrosis is at the core of the high morbidity and mortality rates associated with the complications of diabetes and obesity, including diabetic nephropathy (DN), without any US Food and Drug Administration-approved drugs with this specific target. We recently provided the first evidence that the matricellular protein CCN3 (official symbol NOV) functions in a reciprocal manner, acting on the profibrotic family member CCN2 to inhibit fibrosis in a mesangial cell model of DN. Herein, we used the BT/BR ob/ob mouse as a best model of human obesity and DN progression to determine whether recombinant human CCN3 could be used therapeutically, and the mechanisms involved. Eight weeks of thrice-weekly i.p. injections (0.604 and 6.04 μg/kg of recombinant human CCN3) beginning in early-stage DN completely blocked and/or reversed the up-regulation of mRNA expression of kidney cortex fibrosis genes (CCN2, Col1a2, TGF-β1, and PAI-1) seen in placebo-treated diabetic mice. The treatment completely blocked glomerular fibrosis, as determined by altered mesangial expansion and deposition of laminin. Furthermore, it protected against, or reversed, podocyte loss and kidney function reduction (rise in plasma creatinine concentration); albuminuria was also greatly reduced. This study demonstrates the potential efficacy of recombinant human CCN3 treatment in DN and points to mechanisms operating at multiple levels or pathways, upstream (eg, protecting against cell injury) and downstream (eg, regulating CCN2 activity and extracellular matrix metabolism). PMID:25193594

  18. Regulation of beta catenin signaling and parathyroid hormone anabolic effects in bone by the matricellular protein periostin

    PubMed Central

    Bonnet, Nicolas; Conway, Simon J.; Ferrari, Serge L.

    2012-01-01

    Periostin (Postn) is a matricellular protein preferentially expressed by osteocytes and periosteal osteoblasts in response to mechanical stimulation and parathyroid hormone (PTH). Whether and how periostin expression influences bone anabolism, however, remains unknown. We investigated the skeletal response of adult Postn−/− and Postn+/+ mice to intermittent PTH. Compared with Postn+/+, Postn−/− mice had a lower bone mass, cortical bone volume, and strength response to PTH. PTH-stimulated bone-forming indices were all significantly lower in Postn−/− mice, particularly at the periosteum. Furthermore, in vivo stimulation of Wnt-β-catenin signaling by PTH, as evaluated in TOPGAL reporter mice, was inhibited in the absence of periostin (TOPGAL;Postn−/− mice). PTH stimulated periostin and inhibited MEF2C and sclerostin (Sost) expression in bone and osteoblasts in vitro. Recombinant periostin also suppressed Sost expression, which was mediated through the integrin αVβ3 receptor, whereas periostin-blocking antibody prevented inhibition of MEF2C and Sost by PTH. In turn, administration of a Sost-blocking antiboby partially restored the PTH-mediated increase in bone mass in Postn−/− mice. In addition, primary osteoblasts from Postn−/− mice showed a lower proliferation, mineralization, and migration, both spontaneously and in response to PTH. Osteoblastic gene expression levels confirmed a defect of Postn−/− osteoblast differentiation with and without PTH, as well as an increased osteoblast apoptosis in the absence of periostin. These data elucidate the complex role of periostin on bone anabolism, through the regulation of Sost, Wnt-β-catenin signaling, and osteoblast differentiation. PMID:22927401

  19. Treatment with the matricellular protein CCN3 blocks and/or reverses fibrosis development in obesity with diabetic nephropathy.

    PubMed

    Riser, Bruce L; Najmabadi, Feridoon; Garchow, Kendra; Barnes, Jeffrey L; Peterson, Darryl R; Sukowski, Ernest J

    2014-11-01

    Fibrosis is at the core of the high morbidity and mortality rates associated with the complications of diabetes and obesity, including diabetic nephropathy (DN), without any US Food and Drug Administration-approved drugs with this specific target. We recently provided the first evidence that the matricellular protein CCN3 (official symbol NOV) functions in a reciprocal manner, acting on the profibrotic family member CCN2 to inhibit fibrosis in a mesangial cell model of DN. Herein, we used the BT/BR ob/ob mouse as a best model of human obesity and DN progression to determine whether recombinant human CCN3 could be used therapeutically, and the mechanisms involved. Eight weeks of thrice-weekly i.p. injections (0.604 and 6.04 μg/kg of recombinant human CCN3) beginning in early-stage DN completely blocked and/or reversed the up-regulation of mRNA expression of kidney cortex fibrosis genes (CCN2, Col1a2, TGF-β1, and PAI-1) seen in placebo-treated diabetic mice. The treatment completely blocked glomerular fibrosis, as determined by altered mesangial expansion and deposition of laminin. Furthermore, it protected against, or reversed, podocyte loss and kidney function reduction (rise in plasma creatinine concentration); albuminuria was also greatly reduced. This study demonstrates the potential efficacy of recombinant human CCN3 treatment in DN and points to mechanisms operating at multiple levels or pathways, upstream (eg, protecting against cell injury) and downstream (eg, regulating CCN2 activity and extracellular matrix metabolism).

  20. Tensile stress stimulates the expression of osteogenic cytokines/growth factors and matricellular proteins in the mouse cranial suture at the site of osteoblast differentiation.

    PubMed

    Ikegame, Mika; Tabuchi, Yoshiaki; Furusawa, Yukihiro; Kawai, Mariko; Hattori, Atsuhiko; Kondo, Takashi; Yamamoto, Toshio

    2016-01-01

    Mechanical stress promotes osteoblast proliferation and differentiation from mesenchymal stem cells (MSCs). Although numerous growth factors and cytokines are known to regulate this process, information regarding the differentiation of mechanically stimulated osteoblasts from MSCs in in vivo microenvironment is limited. To determine the significant factors involved in this process, we performed a global analysis of differentially expressed genes, in response to tensile stress, in the mouse cranial suture wherein osteoblasts differentiate from MSCs. We found that the gene expression levels of several components involved in bone morphogenetic protein, Wnt, and epithelial growth factor signalings were elevated with tensile stress. Moreover gene expression of some extracellular matrices (ECMs), such as cysteine rich protein 61 (Cyr61)/CCN1 and galectin-9, were upregulated. These ECMs have the ability to modulate the activities of cytokines and are known as matricellular proteins. Cyr61/CCN1 expression was prominently increased in the fibroblastic cells and preosteoblasts in the suture. Thus, for the first time we demonstrated the mechanical stimulation of Cyr61/CCN1 expression in osteogenic cells in an ex vivo system. These results suggest the importance of matricellular proteins along with the cytokine-mediated signaling for the mechanical regulation of MSC proliferation and differentiation into osteoblastic cell lineage in vivo.

  1. Novel role for matricellular proteins in the regulation of islet β cell survival: the effect of SPARC on survival, proliferation, and signaling.

    PubMed

    Ryall, Claire L; Viloria, Katrina; Lhaf, Fadel; Walker, Anthony J; King, Aileen; Jones, Peter; Mackintosh, David; McNeice, Rosemary; Kocher, Hemant; Flodstrom-Tullberg, Malin; Edling, Charlotte; Hill, Natasha J

    2014-10-31

    Understanding the mechanisms regulating islet growth and survival is critical for developing novel approaches to increasing or sustaining β cell mass in both type 1 and type 2 diabetes patients. Secreted protein acidic and rich in cysteine (SPARC) is a matricellular protein that is important for the regulation of cell growth and adhesion. Increased SPARC can be detected in the serum of type 2 diabetes patients. The aim of this study was to investigate the role of SPARC in the regulation of β cell growth and survival. We show using immunohistochemistry that SPARC is expressed by stromal cells within islets and can be detected in primary mouse islets by Western blot. SPARC is secreted at high levels by pancreatic stellate cells and is regulated by metabolic parameters in these cells, but SPARC expression was not detectable in β cells. In islets, SPARC expression is highest in young mice, and is also elevated in the islets of non-obese diabetic (NOD) mice compared with controls. Purified SPARC inhibits growth factor-induced signaling in both INS-1 β cells and primary mouse islets, and inhibits IGF-1-induced proliferation of INS-1 β cells. Similarly, exogenous SPARC prevents IGF-1-induced survival of primary mouse islet cells. This study identifies the stromal-derived matricellular protein SPARC as a novel regulator of islet survival and β cell growth.

  2. Induction of the matricellular protein CCN1 through RhoA and MRTF-A contributes to ischemic cardioprotection.

    PubMed

    Zhao, Xia; Ding, Eric Y; Yu, Olivia M; Xiang, Sunny Y; Tan-Sah, Valerie P; Yung, Bryan S; Hedgpeth, Joe; Neubig, Richard R; Lau, Lester F; Brown, Joan Heller; Miyamoto, Shigeki

    2014-10-01

    Activation of RhoA, a low molecular-weight G-protein, plays an important role in protecting the heart against ischemic stress. Studies using non-cardiac cells demonstrate that the expression and subsequent secretion of the matricellular protein CCN1 is induced by GPCR agonists that activate RhoA. In this study we determined whether and how CCN1 is induced by GPCR agonists in cardiomyocytes and examined the role of CCN1 in ischemic cardioprotection in cardiomyocytes and the isolated perfused heart. In neonatal rat ventricular myocytes (NRVMs), sphingosine 1-phosphate (S1P), lysophosphatidic acid (LPA) and endothelin-1 induced robust increases in CCN1 expression while phenylephrine, isoproterenol and carbachol had little or no effect. The ability of agonists to activate the small G-protein RhoA correlated with their ability to induce CCN1. CCN1 induction by S1P was blocked when RhoA function was inhibited with C3 exoenzyme or a pharmacological RhoA inhibitor. Conversely overexpression of RhoA was sufficient to induce CCN1 expression. To delineate the signals downstream of RhoA we tested the role of MRTF-A (MKL1), a co-activator of SRF, in S1P-mediated CCN1 expression. S1P increased the nuclear accumulation of MRTF-A and this was inhibited by the functional inactivation of RhoA. In addition, pharmacological inhibitors of MRTF-A or knockdown of MRTF-A significantly diminished S1P-mediated CCN1 expression, indicating a requirement for RhoA/MRTF-A signaling. We also present data indicating that CCN1 is secreted following agonist treatment and RhoA activation, and binds to cells where it can serve an autocrine function. To determine the functional significance of CCN1 expression and signaling, simulated ischemia/reperfusion (sI/R)-induced apoptosis was assessed in NRVMs. The ability of S1P to protect against sI/R was significantly reduced by the inhibition of RhoA, ROCK or MRTF-A or by CCN1 knockdown. We also demonstrate that ischemia/reperfusion induces CCN1 expression

  3. Induction of the matricellular protein CCN1 through RhoA and MRTF-A contributes to ischemic cardioprotection.

    PubMed

    Zhao, Xia; Ding, Eric Y; Yu, Olivia M; Xiang, Sunny Y; Tan-Sah, Valerie P; Yung, Bryan S; Hedgpeth, Joe; Neubig, Richard R; Lau, Lester F; Brown, Joan Heller; Miyamoto, Shigeki

    2014-10-01

    Activation of RhoA, a low molecular-weight G-protein, plays an important role in protecting the heart against ischemic stress. Studies using non-cardiac cells demonstrate that the expression and subsequent secretion of the matricellular protein CCN1 is induced by GPCR agonists that activate RhoA. In this study we determined whether and how CCN1 is induced by GPCR agonists in cardiomyocytes and examined the role of CCN1 in ischemic cardioprotection in cardiomyocytes and the isolated perfused heart. In neonatal rat ventricular myocytes (NRVMs), sphingosine 1-phosphate (S1P), lysophosphatidic acid (LPA) and endothelin-1 induced robust increases in CCN1 expression while phenylephrine, isoproterenol and carbachol had little or no effect. The ability of agonists to activate the small G-protein RhoA correlated with their ability to induce CCN1. CCN1 induction by S1P was blocked when RhoA function was inhibited with C3 exoenzyme or a pharmacological RhoA inhibitor. Conversely overexpression of RhoA was sufficient to induce CCN1 expression. To delineate the signals downstream of RhoA we tested the role of MRTF-A (MKL1), a co-activator of SRF, in S1P-mediated CCN1 expression. S1P increased the nuclear accumulation of MRTF-A and this was inhibited by the functional inactivation of RhoA. In addition, pharmacological inhibitors of MRTF-A or knockdown of MRTF-A significantly diminished S1P-mediated CCN1 expression, indicating a requirement for RhoA/MRTF-A signaling. We also present data indicating that CCN1 is secreted following agonist treatment and RhoA activation, and binds to cells where it can serve an autocrine function. To determine the functional significance of CCN1 expression and signaling, simulated ischemia/reperfusion (sI/R)-induced apoptosis was assessed in NRVMs. The ability of S1P to protect against sI/R was significantly reduced by the inhibition of RhoA, ROCK or MRTF-A or by CCN1 knockdown. We also demonstrate that ischemia/reperfusion induces CCN1 expression

  4. DNA methylation-mediated silencing of matricellular protein dermatopontin promotes hepatocellular carcinoma metastasis by α3β1 integrin-Rho GTPase signaling.

    PubMed

    Fu, Ying; Feng, Ming-Xuan; Yu, Jian; Ma, Ming-Ze; Liu, Xiao-Jin; Li, Jun; Yang, Xiao-Mei; Wang, Ya-Hui; Zhang, Yan-Li; Ao, Jun-Ping; Xue, Feng; Qin, Wenxin; Gu, Jianren; Xia, Qiang; Zhang, Zhi-Gang

    2014-08-30

    Dermatopontin (DPT), a tyrosine-rich, acidic matricellular protein, has been implicated in several human cancers. However, its biological functions and molecular mechanisms in cancer progression, particular hepatocellular carcinoma (HCC), remain unknown. We demonstrated that DPT was significantly down-regulated in 202 HCC clinical samples and that its expression level was closely correlated with cancer metastasis and patient prognosis. The overexpression of DPT dramatically suppressed HCC cell migration in vitro and intrahepatic metastasis in vivo. We further revealed that the down-regulation of DPT in HCC was due to epigenetic silencing by promoter DNA methylation. And the inhibitory effects of DPT on HCC cell motility were associated with dysregulated focal adhesion assembly, decreased RhoA activity and reduced focal adhesion kinase (FAK) and c-Src tyrosine kinase (Src) phosphorylation, and all of these alterations required the involvement of integrin signaling. Furthermore, we determined that the inhibitory effects of DPT on HCC cell motility were primarily mediated through α3β1 integrin. Our study provides new evidence for epigenetic control of tumor microenvironment, and suggests matricellular protein DPT may serve as a novel prognostic marker and act as a HCC metastasis suppressor. PMID:25149533

  5. DNA methylation-mediated silencing of matricellular protein dermatopontin promotes hepatocellular carcinoma metastasis by α3β1 integrin-Rho GTPase signaling.

    PubMed

    Fu, Ying; Feng, Ming-Xuan; Yu, Jian; Ma, Ming-Ze; Liu, Xiao-Jin; Li, Jun; Yang, Xiao-Mei; Wang, Ya-Hui; Zhang, Yan-Li; Ao, Jun-Ping; Xue, Feng; Qin, Wenxin; Gu, Jianren; Xia, Qiang; Zhang, Zhi-Gang

    2014-08-30

    Dermatopontin (DPT), a tyrosine-rich, acidic matricellular protein, has been implicated in several human cancers. However, its biological functions and molecular mechanisms in cancer progression, particular hepatocellular carcinoma (HCC), remain unknown. We demonstrated that DPT was significantly down-regulated in 202 HCC clinical samples and that its expression level was closely correlated with cancer metastasis and patient prognosis. The overexpression of DPT dramatically suppressed HCC cell migration in vitro and intrahepatic metastasis in vivo. We further revealed that the down-regulation of DPT in HCC was due to epigenetic silencing by promoter DNA methylation. And the inhibitory effects of DPT on HCC cell motility were associated with dysregulated focal adhesion assembly, decreased RhoA activity and reduced focal adhesion kinase (FAK) and c-Src tyrosine kinase (Src) phosphorylation, and all of these alterations required the involvement of integrin signaling. Furthermore, we determined that the inhibitory effects of DPT on HCC cell motility were primarily mediated through α3β1 integrin. Our study provides new evidence for epigenetic control of tumor microenvironment, and suggests matricellular protein DPT may serve as a novel prognostic marker and act as a HCC metastasis suppressor.

  6. Receptor communication within the lymphocyte plasma membrane: a role for the thrombospondin family of matricellular proteins.

    PubMed

    Forslöw, A; Liu, Z; Sundqvist, K-G

    2007-01-01

    Lymphocytes, the principal cells of the immune system, carry out immune surveillance throughout the body by their unique capacity to constantly reposition themselves between a free-floating vascular state and a tissue state characterized by migration and frequent adhesive interactions with endothelial cells and components of the extracellular matrix. Therefore, mechanisms co-ordinating adhesion and migration with signals delivered through antigen recognition probably play a pivotal role for the regulation of lymphocyte behaviour and function. Endogenous thrombospondin-1 (TSP-1) seems to be the hub in such a mechanism for autocrine regulation of T cell adhesion and migration. TSP-1 functions as a mediator of cis interaction of vital receptors within the T lymphocyte plasma membrane, including integrins, low density lipoprotein receptor-related protein, calreticulin and integrin-associated protein.

  7. The matricellular protein CCN6 (WISP3) decreases Notch1 and suppresses breast cancer initiating cells.

    PubMed

    Huang, Wei; Martin, Emily E; Burman, Boris; Gonzalez, Maria E; Kleer, Celina G

    2016-05-01

    Increasing evidence supports that the epithelial to mesenchymal transition (EMT) in breast cancer cells generates tumor initiating cells (TICs) but the contribution of the tumor microenvironment to these programs needs further elucidation. CCN6 (WISP3) is a secreted matrix-associated protein (36.9 kDa) of the CCN family (named after CTGF, Cyr61 and Nov) that is reduced or lost in invasive carcinomas of the breast with lymph node metastasis and in inflammatory breast cancer. CCN6 exerts breast cancer growth and invasion inhibitory functions, but the mechanisms remain to be defined. In the present study we discovered that ectopic CCN6 overexpression in triple negative (TN) breast cancer cells and in cells derived from patients is sufficient to induce a mesenchymal to epithelial transition (MET) and to reduce TICs. In vivo, CCN6 overexpression in the TIC population of MDA-MB-231 cells delayed tumor initiation, reduced tumor volume, and inhibited the development of metastasis. Our studies reveal a novel CCN6/Slug signaling axis that regulates Notch1 signaling activation, epithelial cell phenotype and breast TICs, which requires the conserved thrombospondin type 1 (TSP1) motif of CCN6. The relevance of these data to human breast cancer is highlighted by the finding that CCN6 protein levels are inversely correlated with Notch1 intracellular activated form (NICD1) in 69.5% of invasive breast carcinomas. These results demonstrate that CCN6 regulates epithelial and mesenchymal states transition and TIC programs, and pinpoint one responsible mechanism.

  8. The matricellular protein CCN6 (WISP3) decreases Notch1 and suppresses breast cancer initiating cells

    PubMed Central

    Huang, Wei; Martin, Emily E.; Burman, Boris; Gonzalez, Maria E.; Kleer, Celina G.

    2016-01-01

    Increasing evidence supports that the epithelial to mesenchymal transition (EMT) in breast cancer cells generates tumor initiating cells (TICs) but the contribution of the tumor microenvironment to these programs needs further elucidation. CCN6 (WISP3) is a secreted matrix-associated protein (36.9 kDa) of the CCN family (named after CTGF, Cyr61 and Nov) that is reduced or lost in invasive carcinomas of the breast with lymph node metastasis and in inflammatory breast cancer. CCN6 exerts breast cancer growth and invasion inhibitory functions, but the mechanisms remain to be defined. In the present study we discovered that ectopic CCN6 overexpression in triple negative (TN) breast cancer cells and in cells derived from patients is sufficient to induce a mesenchymal to epithelial transition (MET) and to reduce TICs. In vivo, CCN6 overexpression in the TIC population of MDA-MB-231 cells delayed tumor initiation, reduced tumor volume, and inhibited the development of metastasis. Our studies reveal a novel CCN6/Slug signaling axis that regulates Notch1 signaling activation, epithelial cell phenotype and breast TICs, which requires the conserved thrombospondin type 1 (TSP1) motif of CCN6. The relevance of these data to human breast cancer is highlighted by the finding that CCN6 protein levels are inversely correlated with Notch1 intracellular activated form (NICD1) in 69.5% of invasive breast carcinomas. These results demonstrate that CCN6 regulates epithelial and mesenchymal states transition and TIC programs, and pinpoint one responsible mechanism. PMID:26933820

  9. Pleiotropic roles of the matricellular protein Sparc in tendon maturation and ageing

    PubMed Central

    Gehwolf, Renate; Wagner, Andrea; Lehner, Christine; Bradshaw, Amy D.; Scharler, Cornelia; Niestrawska, Justyna A.; Holzapfel, Gerhard A.; Bauer, Hans-Christian; Tempfer, Herbert; Traweger, Andreas

    2016-01-01

    Acute and chronic tendinopathies remain clinically challenging and tendons are predisposed to degeneration or injury with age. Despite the high prevalence of tendon disease in the elderly, our current understanding of the mechanisms underlying the age-dependent deterioration of tendon function remains very limited. Here, we show that Secreted protein acidic and rich in cysteine (Sparc) expression significantly decreases in healthy-aged mouse Achilles tendons. Loss of Sparc results in tendon collagen fibrillogenesis defects and Sparc−/− tendons are less able to withstand force in comparison with their respective wild type counterparts. On the cellular level, Sparc-null and healthy-aged tendon-derived cells exhibited a more contracted phenotype and an altered actin cytoskeleton. Additionally, an elevated expression of the adipogenic marker genes PPARγ and Cebpα with a concomitant increase in lipid deposits in aged and Sparc−/− tendons was observed. In summary, we propose that Sparc levels in tendons are critical for proper collagen fibril maturation and its age-related decrease, together with a change in ECM properties favors lipid accretion in tendons. PMID:27586416

  10. Pleiotropic roles of the matricellular protein Sparc in tendon maturation and ageing.

    PubMed

    Gehwolf, Renate; Wagner, Andrea; Lehner, Christine; Bradshaw, Amy D; Scharler, Cornelia; Niestrawska, Justyna A; Holzapfel, Gerhard A; Bauer, Hans-Christian; Tempfer, Herbert; Traweger, Andreas

    2016-01-01

    Acute and chronic tendinopathies remain clinically challenging and tendons are predisposed to degeneration or injury with age. Despite the high prevalence of tendon disease in the elderly, our current understanding of the mechanisms underlying the age-dependent deterioration of tendon function remains very limited. Here, we show that Secreted protein acidic and rich in cysteine (Sparc) expression significantly decreases in healthy-aged mouse Achilles tendons. Loss of Sparc results in tendon collagen fibrillogenesis defects and Sparc-/- tendons are less able to withstand force in comparison with their respective wild type counterparts. On the cellular level, Sparc-null and healthy-aged tendon-derived cells exhibited a more contracted phenotype and an altered actin cytoskeleton. Additionally, an elevated expression of the adipogenic marker genes PPARγ and Cebpα with a concomitant increase in lipid deposits in aged and Sparc-/- tendons was observed. In summary, we propose that Sparc levels in tendons are critical for proper collagen fibril maturation and its age-related decrease, together with a change in ECM properties favors lipid accretion in tendons. PMID:27586416

  11. Pleiotropic roles of the matricellular protein Sparc in tendon maturation and ageing.

    PubMed

    Gehwolf, Renate; Wagner, Andrea; Lehner, Christine; Bradshaw, Amy D; Scharler, Cornelia; Niestrawska, Justyna A; Holzapfel, Gerhard A; Bauer, Hans-Christian; Tempfer, Herbert; Traweger, Andreas

    2016-09-02

    Acute and chronic tendinopathies remain clinically challenging and tendons are predisposed to degeneration or injury with age. Despite the high prevalence of tendon disease in the elderly, our current understanding of the mechanisms underlying the age-dependent deterioration of tendon function remains very limited. Here, we show that Secreted protein acidic and rich in cysteine (Sparc) expression significantly decreases in healthy-aged mouse Achilles tendons. Loss of Sparc results in tendon collagen fibrillogenesis defects and Sparc-/- tendons are less able to withstand force in comparison with their respective wild type counterparts. On the cellular level, Sparc-null and healthy-aged tendon-derived cells exhibited a more contracted phenotype and an altered actin cytoskeleton. Additionally, an elevated expression of the adipogenic marker genes PPARγ and Cebpα with a concomitant increase in lipid deposits in aged and Sparc-/- tendons was observed. In summary, we propose that Sparc levels in tendons are critical for proper collagen fibril maturation and its age-related decrease, together with a change in ECM properties favors lipid accretion in tendons.

  12. Matricellular protein CCN1/CYR61: a new player in inflammation and leukocyte trafficking.

    PubMed

    Emre, Yalin; Imhof, Beat A

    2014-03-01

    Cystein-rich protein 61 (CYR61/CCN1) is a component of the extracellular matrix, which is produced and secreted by several cell types including endothelial cells, fibroblasts and smooth muscle cells. CCN1 has been implicated in leukocyte migration and the inflammatory process, but it is also involved in cardiovascular development and carcinogenesis. It exerts its functions through binding to multiple integrins present in many different cell types. This multiplicity in function is now known to contribute to the diverse array of cellular processes it can regulate. The expression of CCN1 is tightly regulated by cytokines and growth factors. However, CCN1 can directly modulate cell adhesion and migratory processes whilst simultaneously regulating the production of other cytokines and chemokines through paracrine and autocrine feedback loops. This complex functionality of CCN1 has highlighted the pivotal role this molecule can play in regulating the immunosurveillance process. Furthermore, CCN1 has now emerged as an important partner when targeting components of the infectious or chronic inflammatory disease processes such as atherosclerosis or rheumatoid arthritis. This review will focus on CYR61/CCN1 and its ability to control the migration of leukocytes, the production of cytokines and cell proliferation or senescence at the site of inflammation.

  13. Matricellular protein thrombospondins: influence on ocular angiogenesis, wound healing and immuneregulation.

    PubMed

    Masli, Sharmila; Sheibani, Nader; Cursiefen, Claus; Zieske, James

    2014-08-01

    Thrombospondins are a family of large multi-domain glycoproteins described as matricelluar proteins based on their ability to interact with a broad range of receptors, matrix molecules, growth factors or proteases, and to modulate array of cellular functions including intracellular signaling, proliferation and migration. Two members of the thrombospondin family, thrombospondin 1 (TSP-1) and thrombospondin 2 (TSP-2) are studied extensively to determine their structure and function. While expressed at low levels in normal adult tissues, their increased expression is seen predominantly in response to cellular perturbations. Despite structural similarities, a notable functional difference between TSP-1 and TSP-2 includes the ability of former to activate of latent TGF-β and its competitive inhibition by the latter. Both these thrombospondins are reported to play important roles in TGF-β rich ocular environment with most reports related to TSP-1. They are expressed by many ocular cell types and detectable in the aqueous and vitreous humor. TSP-1 and TSP-2 influence many cellular interactions in the eye such as angiogenesis, cell migration, wound healing, TGF-β activation and regulation of inflammatory immune responses. Together, these processes are known to contribute to the immune privilege status of the eye. Emerging roles of TSP-1 and TSP-2 in ocular functions and pathology are reviewed here.

  14. Role of β-catenin-regulated CCN matricellular proteins in epithelial repair after inflammatory lung injury.

    PubMed

    Zemans, Rachel L; McClendon, Jazalle; Aschner, Yael; Briones, Natalie; Young, Scott K; Lau, Lester F; Kahn, Michael; Downey, Gregory P

    2013-03-15

    Repair of the lung epithelium after injury is integral to the pathogenesis and outcomes of diverse inflammatory lung diseases. We previously reported that β-catenin signaling promotes epithelial repair after inflammatory injury, but the β-catenin target genes that mediate this effect are unknown. Herein, we examined which β-catenin transcriptional coactivators and target genes promote epithelial repair after inflammatory injury. Transmigration of human neutrophils across cultured monolayers of human lung epithelial cells resulted in a fall in transepithelial resistance and the formation of discrete areas of epithelial denudation ("microinjury"), which repaired via cell spreading by 96 h. In mice treated with intratracheal (i.t.) LPS or keratinocyte chemokine, neutrophil emigration was associated with increased permeability of the lung epithelium, as determined by increased bronchoalveolar lavage (BAL) fluid albumin concentration, which decreased over 3-6 days. Activation of β-catenin/p300-dependent gene expression using the compound ICG-001 accelerated epithelial repair in vitro and in murine models. Neutrophil transmigration induced epithelial expression of the β-catenin/p300 target genes Wnt-induced secreted protein (WISP) 1 and cysteine-rich (Cyr) 61, as determined by real-time PCR (qPCR) and immunostaining. Purified neutrophil elastase induced WISP1 upregulation in lung epithelial cells, as determined by qPCR. WISP1 expression increased in murine lungs after i.t. LPS, as determined by ELISA of the BAL fluid and qPCR of whole lung extracts. Finally, recombinant WISP1 and Cyr61 accelerated repair, and Cyr61-neutralizing antibodies delayed repair of the injured epithelium in vitro. We conclude that β-catenin/p300-dependent expression of WISP1 and Cyr61 is critical for epithelial repair and represents a potential therapeutic target to promote epithelial repair after inflammatory injury.

  15. Integrin binding and mechanical tension induce movement of mRNA and ribosomes to focal adhesions

    NASA Technical Reports Server (NTRS)

    Chicurel, M. E.; Singer, R. H.; Meyer, C. J.; Ingber, D. E.

    1998-01-01

    The extracellular matrix (ECM) activates signalling pathways that control cell behaviour by binding to cell-surface integrin receptors and inducing the formation of focal adhesion complexes (FACs). In addition to clustered integrins, FACs contain proteins that mechanically couple the integrins to the cytoskeleton and to immobilized signal-transducing molecules. Cell adhesion to the ECM also induces a rapid increase in the translation of preexisting messenger RNAs. Gene expression can be controlled locally by targeting mRNAs to specialized cytoskeletal domains. Here we investigate whether cell binding to the ECM promotes formation of a cytoskeletal microcompartment specialized for translational control at the site of integrin binding. High-resolution in situ hybridization revealed that mRNA and ribosomes rapidly and specifically localized to FACs that form when cells bind to ECM-coated microbeads. Relocation of these protein synthesis components to the FAC depended on the ability of integrins to mechanically couple the ECM to the contractile cytoskeleton and on associated tension-moulding of the actin lattice. Our results suggest a new type of gene regulation by integrins and by mechanical stress which may involve translation of mRNAs into proteins near the sites of signal reception.

  16. Sustained augmentation of cardiac alpha1A-adrenergic drive results in pathological remodeling with contractile dysfunction, progressive fibrosis and reactivation of matricellular protein genes.

    PubMed

    Chaulet, H; Lin, F; Guo, J; Owens, W A; Michalicek, J; Kesteven, S H; Guan, Z; Prall, O W; Mearns, B M; Feneley, M P; Steinberg, S F; Graham, R M

    2006-04-01

    We previously reported that transgenic (TG) mice with cardiac-restricted alpha(1A)-adrenergic receptor (alpha(1A)-AR)-overexpression showed enhanced contractility, but no hypertrophy. Since chronic inotropic enhancement may be deleterious, we investigated if long-term, cardiac function and longevity are compromised. alpha(1A)-TG mice, but not their non-TG littermates (NTLs), showed progressive loss of left ventricular (LV) hypercontractility (dP/dt(max): 14,567+/-603 to 11,610+/-915 mmHg/s, P<0.05, A1A1 line: 170-fold overexpression; and 13,625+/-826 to 8322+/-682 mmHg/s, respectively, P<0.05, A1A4 line: 112-fold overexpression, at 2 and 6 months, respectively). Both TG lines developed LV fibrosis, but not LV dilatation or hypertrophy, despite activation of hypertrophic signaling pathways. Microarray and real time RT-PCR analyses revealed activation of matricellular protein genes, including those for thrombospondin 1, connective tissue growth factor and tenascin C, but not transforming growth factor beta1. Life-span was markedly shortened (mean age at death: 155 days, A1A1 line; 224 days, A1A4 line compared with NTLs: >300 days). Telemetric electrocardiography revealed that death in the alpha(1A)-AR TG mice was due to cardiac standstill preceded by a progressive diminution in QRS amplitude, but not by arrhythmias. The QRS changes and sudden death could be mimicked by alpha(1)-AR activation, and reversed preterminally by alpha(1)-AR blockade, suggesting a relationship to stress- or activity-associated catecholamine release. Thus, long-term augmentation of cardiac alpha(1A)-adrenergic drive leads to premature death and progressive LV fibrosis with reactivation of matricellular protein genes. To our knowledge this is the first evidence in vivo for a role of the alpha(1A)-AR in ventricular fibrosis and in pathological cardiac remodeling.

  17. Osteopontin Is Cleaved at Multiple Sites Close to Its Integrin-binding Motifs in Milk and Is a Novel Substrate for Plasmin and Cathepsin D*

    PubMed Central

    Christensen, Brian; Schack, Lotte; Kläning, Eva; Sørensen, Esben S.

    2010-01-01

    Osteopontin (OPN) is a highly modified integrin-binding protein present in most tissues and body fluids where it has been implicated in numerous biological processes. A significant regulation of OPN function is mediated through phosphorylation and proteolytic processing. Proteolytic cleavage by thrombin and matrix metalloproteinases close to the integrin-binding Arg-Gly-Asp sequence modulates the function of OPN and its integrin binding properties. In this study, seven N-terminal OPN fragments originating from proteolytic cleavage have been characterized from human milk. Identification of the cleavage sites revealed that all fragments contained the Arg–Gly–Asp145 sequence and were generated by cleavage of the Leu151–Arg152, Arg152–Ser153, Ser153–Lys154, Lys154–Ser155, Ser155–Lys156, Lys156–Lys157, or Phe158–Arg159 peptide bonds. Six cleavages cannot be ascribed to thrombin or matrix metalloproteinase activity, whereas the cleavage at Arg152–Ser153 matches thrombin specificity for OPN. The principal protease in milk, plasmin, hydrolyzed the same peptide bond as thrombin, but its main cleavage site was identified to be Lys154–Ser155. Another endogenous milk protease, cathepsin D, cleaved the Leu151–Arg152 bond. OPN fragments corresponding to plasmin activity were also identified in urine showing that plasmin cleavage of OPN is not restricted to milk. Plasmin, but not cathepsin D, cleavage of OPN increased cell adhesion mediated by the αVβ3- or α5β1-integrins. Similar cellular adhesion was mediated by plasmin and thrombin-cleaved OPN showing that plasmin can be a potent regulator of OPN activity. These data show that OPN is highly susceptible to cleavage near its integrin-binding motifs, and the protein is a novel substrate for plasmin and cathepsin D. PMID:20071328

  18. Expression of mRNA of apolipoprotein E, apolipoprotein A-IV, and matricellular proteins in the myocardium and intensity of fibroplastic processes during experimental hypercholesterolemia.

    PubMed

    Lushnikova, E L; Nepomnyashchikh, L M; Pichigin, V I; Klinnikova, M G; Nepomnyashchikh, R D; Sergeevichev, D S

    2013-12-01

    The expression of mRNA of matricellular proteins (osteopontin, and lumican), apolipoproteins E and A-IV, and microsomal triglyceride transfer protein, and the intensity of fibroplastic processes were studied in the myocardium of rats during experimental chronic hypercholesterolemia. We have found that the development of chronic hypercholesterolemia was followed by an increase in volume density of interstitial connective tissue in the myocardium reflecting the activation of fibroplastic processes. A slight positive correlation was observed between the connective tissue density in the myocardium and expression of osteopontin mRNA (r=0.408) and lumican mRNA (r=0.470). Myocardium remodeling during hypercholesterolemia is realized against the background of increased expression of apolipoproteins E and A-IV mRNA and microsomal triglyceride transfer protein mRNA involved in transport and metabolism of lipoproteins in several tissues and probably play a pivotal role in the regulation of lipoprotein transport and metabolism in the myocardium. We concluded that the increase in the expression of apolipoproteins (E and A-IV) and microsomal triglyceride transfer protein play adaptive and compensatory role and is related to the increase in lipoprotein utilization by macrophages.

  19. Collagen matrix-induced expression of integrin αVβ3 in circulating angiogenic cells can be targeted by matricellular protein CCN1 to enhance their function.

    PubMed

    McNeill, Brian; Vulesevic, Branka; Ostojic, Aleksandra; Ruel, Marc; Suuronen, Erik J

    2015-04-01

    Circulating angiogenic cells (CACs) play an important role in vascular homeostasis and hold therapeutic promise for treating a variety of cardiovascular diseases. However, further improvements are needed because the effects of CAC therapy remain minimal or transient. The regenerative potential of these cells can be improved by culture on a collagen-based matrix through the up-regulation of key integrin proteins. We found that human CAC function was enhanced by using the matricellular protein CCN1 (CYR61/CTGF/NOV family member 1) to target integrin αV and β3, which are up-regulated on matrix. Compared to matrix-cultured CACs, CCN1-matrix CACs exhibited a 2.2-fold increase in cell proliferation, 1.8-fold greater migration toward VEGF, and 1.7-fold more incorporation into capillary-like structures in an angiogenesis assay. In vivo, intramuscular injection of CCN1-matrix-cultured CACs into ischemic hind limbs of CD-1 nude mice resulted in blood flow recovery to 80% of baseline, which was greater than matrix-cultured CACs (66%) and PBS (35%) treatment groups. Furthermore, transplanted CCN1-matrix CACs exhibited greater engraftment (11-fold) and stimulated the up-regulation of survival and angiogenic genes (>3-fold). These findings reveal the importance of cell-matrix interactions in regulating CAC function and also reveal a mechanism by which these may be exploited to enhance cell therapies for ischemic disease.

  20. Balanced regulation of the CCN family of matricellular proteins: a novel approach to the prevention and treatment of fibrosis and cancer.

    PubMed

    Riser, Bruce L; Barnes, Jeffrey L; Varani, James

    2015-12-01

    The CCN family of matricellular signaling proteins is emerging as a unique common link across multiple diseases and organs related to injury and repair. They are now being shown to play a central role in regulating the pathways to the initiation and resolution of normal wound healing and fibrosis in response to multiple forms of injury. Similarly, it is also emerging that they play a key role in regulating the establishment, growth, metastases and tissue regeneration in many forms of cancer via the interaction of cancer cells with the tumor stroma. Evidence has been recently provided that these proteins do not act independently but are co-regulated working in a yin/yang manner to alter the outcome of both normal physiological processes as well as pathology. The purpose of this review is to twofold. First, it will summarize work to date supporting CCN2 as a therapeutic target in the formation and progression of renal, skin, and other organ fibrosis, as well as cancer stroma formation. Second, it will highlight recent evidence for CCN3 as a counter-regulator and a potential therapeutic agent in these diseases with an exciting, novel potential to both treat and then restore tissue homeostasis in those afflicted by these devastating disorders.

  1. Molecular cloning and characterization of the matricellular protein Sparc/osteonectin in flatfish, Scophthalmus maximus, and its developmental stage-dependent transcriptional regulation during metamorphosis.

    PubMed

    Torres-Núñez, E; Suarez-Bregua, P; Cal, L; Cal, R; Cerdá-Reverter, J M; Rotllant, J

    2015-09-01

    SPARC/osteonectin is a multifunctional matricellular glycoprotein, which is expressed in embryonic and adult tissues that undergo active proliferation and dynamic morphogenesis. Recent studies indicate that Sparc expression appears early in development, although its function and regulation during development are largely unknown. In this report, we describe the isolation, characterization, post-embryonic developmental expression and environmental thermal regulation of sparc in turbot. The full-length turbot sparc cDNA contains 930 bp and encodes a protein of 310 amino acids, which shares 77, 75 and 80% identity with human, frog and zebrafish, respectively. Results of whole-mount in situ hybridization reveal a dynamic expression profile during post-embryonic turbot development. Sparc is expressed differentially in the cranioencephalic region; mainly in jaws, branchial arches, fin folds and rays of caudal, dorsal and anal fins. Furthermore, ontogenetic studies demonstrated that Sparc gene expression is dynamically regulated during post-embryonic turbot development, with high expression during stage-specific post-embryonic remodeling. Additionally, the effect of thermal environmental conditions on turbot development and on ontogenetic sparc expression was evaluated.

  2. Astrocyte matricellular proteins that control excitatory synaptogenesis are regulated by inflammatory cytokines and correlate with paralysis severity during experimental autoimmune encephalomyelitis.

    PubMed

    Blakely, Pennelope K; Hussain, Shabbir; Carlin, Lindsey E; Irani, David N

    2015-01-01

    The matricellular proteins, secreted protein acidic and rich in cysteine (SPARC) and SPARC-like 1 (SPARCL1), are produced by astrocytes and control excitatory synaptogenesis in the central nervous system. While SPARCL1 directly promotes excitatory synapse formation in vitro and in the developing nervous system in vivo, SPARC specifically antagonizes the synaptogenic actions of SPARCL1. We hypothesized these proteins also help maintain existing excitatory synapses in adult hosts, and that local inflammation in the spinal cord alters their production in a way that dynamically modulates motor synapses and impacts the severity of paralysis during experimental autoimmune encephalomyelitis (EAE) in mice. Using a spontaneously remitting EAE model, paralysis severity correlated inversely with both expression of synaptic proteins and the number of synapses in direct contact with the perikarya of motor neurons in spinal gray matter. In both remitting and non-remitting EAE models, paralysis severity also correlated inversely with sparcl1:sparc transcript and SPARCL1:SPARC protein ratios directly in lumbar spinal cord tissue. In vitro, astrocyte production of both SPARCL1 and SPARC was regulated by T cell-derived cytokines, causing dynamic modulation of the SPARCL1:SPARC expression ratio. Taken together, these data support a model whereby proinflammatory cytokines inhibit SPARCL1 and/or augment SPARC expression by astrocytes in spinal gray matter that, in turn, cause either transient or sustained synaptic retraction from lumbar spinal motor neurons thereby regulating hind limb paralysis during EAE. Ongoing studies seek ways to alter this SPARCL1:SPARC expression ratio in favor of synapse reformation/maintenance and thus help to modulate neurologic deficits during times of inflammation. This could identify new astrocyte-targeted therapies for diseases such as multiple sclerosis. PMID:26500475

  3. Astrocyte matricellular proteins that control excitatory synaptogenesis are regulated by inflammatory cytokines and correlate with paralysis severity during experimental autoimmune encephalomyelitis.

    PubMed

    Blakely, Pennelope K; Hussain, Shabbir; Carlin, Lindsey E; Irani, David N

    2015-01-01

    The matricellular proteins, secreted protein acidic and rich in cysteine (SPARC) and SPARC-like 1 (SPARCL1), are produced by astrocytes and control excitatory synaptogenesis in the central nervous system. While SPARCL1 directly promotes excitatory synapse formation in vitro and in the developing nervous system in vivo, SPARC specifically antagonizes the synaptogenic actions of SPARCL1. We hypothesized these proteins also help maintain existing excitatory synapses in adult hosts, and that local inflammation in the spinal cord alters their production in a way that dynamically modulates motor synapses and impacts the severity of paralysis during experimental autoimmune encephalomyelitis (EAE) in mice. Using a spontaneously remitting EAE model, paralysis severity correlated inversely with both expression of synaptic proteins and the number of synapses in direct contact with the perikarya of motor neurons in spinal gray matter. In both remitting and non-remitting EAE models, paralysis severity also correlated inversely with sparcl1:sparc transcript and SPARCL1:SPARC protein ratios directly in lumbar spinal cord tissue. In vitro, astrocyte production of both SPARCL1 and SPARC was regulated by T cell-derived cytokines, causing dynamic modulation of the SPARCL1:SPARC expression ratio. Taken together, these data support a model whereby proinflammatory cytokines inhibit SPARCL1 and/or augment SPARC expression by astrocytes in spinal gray matter that, in turn, cause either transient or sustained synaptic retraction from lumbar spinal motor neurons thereby regulating hind limb paralysis during EAE. Ongoing studies seek ways to alter this SPARCL1:SPARC expression ratio in favor of synapse reformation/maintenance and thus help to modulate neurologic deficits during times of inflammation. This could identify new astrocyte-targeted therapies for diseases such as multiple sclerosis.

  4. The matricellular "cysteine-rich protein 61" is released from activated platelets and increased in the circulation during experimentally induced sepsis.

    PubMed

    Hviid, Claus Vinter B; Samulin Erdem, Johanna; Drechsler, Susanne; Weixelbaumer, Katrin; Ahmed, M Shakil; Attramadal, Håvard; Redl, Heinz; Bahrami, Soheyl; Osuchowski, Marcin F; Aasen, Ansgar O

    2014-03-01

    Sepsis and sepsis-induced organ dysfunction remain lethal and common conditions among intensive care patients. Accumulating evidence suggests that the matricellular Cyr61/CCN1 (cysteine-rich, angiogenic-inducer, 61) protein is involved in the regulation of inflammatory responses and possesses organ-protective capabilities in diseases of an inflammatory etiology. However, its regulation in sepsis remains largely unexplored. The present study provides a comprehensive description of CCN1 regulation in the circulation and vital organs during experimentally induced sepsis with developing organ dysfunction. Female CD-1 mice served as baseline controls or were subjected to cecal ligation and puncture (CLP) for 18 to 96 h, and CCN1 regulation was analyzed in selected organs and in the circulation. A 5-, 5-, and 3-fold increases in circulating CCN1 protein were observed at 18, 48, and 96 h after CLP, respectively. Hepatic and pulmonary CCN1 mRNA expression was down-regulated by 80%, 60%, and 55% and 85%, 80%, and 65% at 18, 48, and 96 h after CLP and undetectable in circulating white blood cells. To identify a potential source for the circulating protein, mouse and human platelets were explored and revealed to contain CCN1. Human platelets were stimulated by thrombin and a specific PAR1 agonist (SFLLRN) in vitro. Both agonists induced an instant CCN1 release, and the effect of SFLLRN was blocked by the specific antagonist RWJ56110. The current study demonstrates that experimental sepsis is associated with a robust increase in circulating CCN1 protein levels and a paradoxical downregulation of CCN1 mRNA expression in vital organs. It provides evidence that CCN1 is released from activated platelets, suggesting that platelets constitute a novel source for CCN1 release to the circulation during sepsis. PMID:24430538

  5. The matricellular "cysteine-rich protein 61" is released from activated platelets and increased in the circulation during experimentally induced sepsis.

    PubMed

    Hviid, Claus Vinter B; Samulin Erdem, Johanna; Drechsler, Susanne; Weixelbaumer, Katrin; Ahmed, M Shakil; Attramadal, Håvard; Redl, Heinz; Bahrami, Soheyl; Osuchowski, Marcin F; Aasen, Ansgar O

    2014-03-01

    Sepsis and sepsis-induced organ dysfunction remain lethal and common conditions among intensive care patients. Accumulating evidence suggests that the matricellular Cyr61/CCN1 (cysteine-rich, angiogenic-inducer, 61) protein is involved in the regulation of inflammatory responses and possesses organ-protective capabilities in diseases of an inflammatory etiology. However, its regulation in sepsis remains largely unexplored. The present study provides a comprehensive description of CCN1 regulation in the circulation and vital organs during experimentally induced sepsis with developing organ dysfunction. Female CD-1 mice served as baseline controls or were subjected to cecal ligation and puncture (CLP) for 18 to 96 h, and CCN1 regulation was analyzed in selected organs and in the circulation. A 5-, 5-, and 3-fold increases in circulating CCN1 protein were observed at 18, 48, and 96 h after CLP, respectively. Hepatic and pulmonary CCN1 mRNA expression was down-regulated by 80%, 60%, and 55% and 85%, 80%, and 65% at 18, 48, and 96 h after CLP and undetectable in circulating white blood cells. To identify a potential source for the circulating protein, mouse and human platelets were explored and revealed to contain CCN1. Human platelets were stimulated by thrombin and a specific PAR1 agonist (SFLLRN) in vitro. Both agonists induced an instant CCN1 release, and the effect of SFLLRN was blocked by the specific antagonist RWJ56110. The current study demonstrates that experimental sepsis is associated with a robust increase in circulating CCN1 protein levels and a paradoxical downregulation of CCN1 mRNA expression in vital organs. It provides evidence that CCN1 is released from activated platelets, suggesting that platelets constitute a novel source for CCN1 release to the circulation during sepsis.

  6. Novel adhesin from Pasteurella multocida that binds to the integrin-binding fibronectin FnIII9-10 repeats.

    PubMed

    Mullen, Lisa M; Nair, Sean P; Ward, John M; Rycroft, Andrew N; Williams, Rachel J; Robertson, Giles; Mordan, Nicky J; Henderson, Brian

    2008-03-01

    Phage display screening with fragmented genomic DNA from the animal pathogen Pasteurella multocida has identified a gene encoding a putative fibronectin binding protein (19). Homologues of this gene (PM1665) are found in all other sequenced members of the Pasteurellaceae. Gene PM1665 has been cloned, and the protein has been expressed. Recombinant PM1665 protein binds to both soluble and immobilized fibronectin and is unique in that it interacts with the integrin-binding fibronectin type III (FnIII) repeats FnIII(9-10) and not, as is the case for almost all other fibronectin adhesins, to the N-terminal type I repeats. Surface plasmon resonance analysis revealed a complex binding mechanism with a K(D) (equilibrium dissociation constant) of 150 nM +/- 70 nM. Bioinformatics analysis suggests that the PM1665 protein contains two helix-hairpin-helix (HhH) motifs, and truncation mutation studies have identified the binding site in the protein as a combination of these two HhH motifs in conjunction with a conserved amino acid motif, VNINTA. We have shown that the PM1665 protein is on the cell surface and that binding of P. multocida to fibronectin is almost completely inhibited by anti-PM1665 antiserum. These results support the hypothesis that the PM1665 protein is a member of a new family of fibronectin binding adhesins that are important in the adhesion of P. multocida to fibronectin. PMID:18160478

  7. The matri-cellular proteins 'cysteine-rich, angiogenic-inducer, 61' and 'connective tissue growth factor' are regulated in experimentally-induced sepsis with multiple organ dysfunction.

    PubMed

    Hviid, Claus V B; Erdem, Johanna Samulin; Kunke, David; Ahmed, Shakil M; Kjeldsen, Signe F; Wang, Yun Yong; Attramadal, Håvard; Aasen, Ansgar O

    2012-10-01

    Organ failure is a severe complication in sepsis for which the pathophysiology remains incompletely understood. Recently, the matri-cellular cysteine-rich, angiogenic induced, 61 (Cyr61/CCN1); connective tissue growth factor (Ctgf/CCN2); and nephroblastoma overexpressed gene (Nov/CCN3) (CCN)-protein family have been attributed organ-protective properties. Their expression is sensitive to mediators of sepsis pathophysiology but a potential role in sepsis remains elusive. To provide an initial assessment, 50 rats were subjected to 18 h of cecal-ligation and puncture or sham operation. Hepatic and pulmonary CCN1 mRNA displayed an average 7.4- and 3.3-fold induction, while its cardiac expression was unchanged. The changes coincided with excessive hepatic and pulmonary inflammatory gene activation and a restricted cardiac inflammation. Furthermore, hepatocytes displayed a dosage-dependent CCN1 mRNA response in vitro, supporting a cytokine-mediated CCN1 regulation in sepsis. CCN2 mRNA was 2.2-fold induced in the liver, while 2.0-fold and 1.4-fold repressed in the heart and lung. Meanwhile, it did not respond to TNF-α exposure in vitro, which indicates different means of regulation than for CCN1. Taken together, this study provides the first evidence for multi-organ regulation of CCN1 and CCN2 in early stages of sepsis, and implies the eruption of inflammatory mediators as a potential mechanism behind the observed CCN1 regulation.

  8. IFATS collection: Combinatorial peptides identify alpha5beta1 integrin as a receptor for the matricellular protein SPARC on adipose stromal cells.

    PubMed

    Nie, Jing; Chang, Benny; Traktuev, Dmitry O; Sun, Jessica; March, Keith; Chan, Lawrence; Sage, E Helene; Pasqualini, Renata; Arap, Wadih; Kolonin, Mikhail G

    2008-10-01

    The biological features of adipose stromal (stem) cells (ASC), which serve as progenitors for differentiated cells of white adipose tissue (WAT), are still largely undefined. In an initiative to identify functional ASC surface receptors, we screened a combinatorial library for peptide ligands binding to patient-derived ASC. We demonstrate that both primary and cultured human and mouse stromal cells express a conserved receptor targeted by peptides found to mimic SPARC, a matricellular protein that is required for normal WAT development. A signaling receptor for SPARC has not as yet been determined. By using the SPARC-mimicking peptides CMLAGWIPC (termed hPep) and CWLGEWLGC (termed mPep), isolated by panning on human and mouse cells, respectively, we identified the alpha5beta1 integrin complex as a candidate receptor for SPARC. On the basis of these results, we evaluated ASC responses to SPARC or SPARC-mimicking peptide exposure. Our results suggest that extracellular SPARC binds to alpha5beta1 integrin at sites of focal adhesions, an interaction disrupting firm attachment of ASC to extracellular matrix. We propose that SPARC-mediated mobilization of ASC through its effect on alpha5beta1 integrin complex provides a functional basis for the regulation of WAT body composition by SPARC. We also show that alpha5beta1 integrin is a potential target for ASC-selective intracellular delivery of bioactive peptides and gene therapy vectors directed by the SPARC-mimicking peptides. Disclosure of potential conflicts of interest is found at the end of this article.

  9. Crystal structure of vinculin in complex with vinculin binding site 50 (VBS50), the integrin binding site 2 (IBS2) of talin

    SciTech Connect

    Yogesha, S.D.; Rangarajan, Erumbi S.; Vonrhein, Clemens; Bricogne, Gerard; Izard, Tina

    2012-05-10

    The cytoskeletal protein talin activates integrin receptors by binding of its FERM domain to the cytoplasmic tail of {beta}-integrin. Talin also couples integrins to the actin cytoskeleton, largely by binding to and activating the cytoskeletal protein vinculin, which binds to F-actin through the agency of its five-helix bundle tail (Vt) domain. Talin activates vinculin by means of buried amphipathic {alpha}-helices coined vinculin binding sites (VBSs) that reside within numerous four- and five-helix bundle domains that comprise the central talin rod, which are released from their buried locales by means of mechanical tension on the integrin:talin complex. In turn, these VBSs bind to the N-terminal seven-helix bundle (Vh1) domain of vinculin, creating an entirely new helix bundle that severs its head-tail interactions. Interestingly, talin harbors a second integrin binding site coined IBS2 that consists of two five-helix bundle domains that also contain a VBS (VBS50). Here we report the crystal structure of VBS50 in complex with vinculin at 2.3 {angstrom} resolution and show that intramolecular interactions of VBS50 within IBS2 are much more extensive versus its interactions with vinculin. Indeed, the IBS2-vinculin interaction only occurs at physiological temperature and the affinity of VBS50 for vinculin is about 30 times less than other VBSs. The data support a model where integrin binding destabilizes IBS2 to allow it to bind to vinculin.

  10. Mimicking bone extracellular matrix: integrin-binding peptidomimetics enhance osteoblast-like cells adhesion, proliferation, and differentiation on titanium.

    PubMed

    Fraioli, Roberta; Rechenmacher, Florian; Neubauer, Stefanie; Manero, José M; Gil, Javier; Kessler, Horst; Mas-Moruno, Carlos

    2015-04-01

    Interaction between the surface of implants and biological tissues is a key aspect of biomaterials research. Apart from fulfilling the non-toxicity and structural requirements, synthetic materials are asked to direct cell response, offering engineered cues that provide specific instructions to cells. This work explores the functionalization of titanium with integrin-binding peptidomimetics as a novel and powerful strategy to improve the adhesion, proliferation and differentiation of osteoblast-like cells to implant materials. Such biomimetic strategy aims at targeting integrins αvβ3 and α5β1, which are highly expressed on osteoblasts and are essential for many fundamental functions in bone tissue development. The successful grafting of the bioactive molecules on titanium is proven by contact angle measurements, X-ray photoelectron spectroscopy and fluorescent labeling. Early attachment and spreading of cells are statistically enhanced by both peptidomimetics compared to unmodified titanium, reaching values of cell adhesion comparable to those obtained with full-length extracellular matrix proteins. Moreover, an increase in alkaline phosphatase activity, and statistically higher cell proliferation and mineralization are observed on surfaces coated with the peptidomimetics. This study shows an unprecedented biological activity for low-molecular-weight ligands on titanium, and gives striking evidence of the potential of these molecules to foster bone regeneration on implant materials. PMID:25637448

  11. Mimicking bone extracellular matrix: integrin-binding peptidomimetics enhance osteoblast-like cells adhesion, proliferation, and differentiation on titanium.

    PubMed

    Fraioli, Roberta; Rechenmacher, Florian; Neubauer, Stefanie; Manero, José M; Gil, Javier; Kessler, Horst; Mas-Moruno, Carlos

    2015-04-01

    Interaction between the surface of implants and biological tissues is a key aspect of biomaterials research. Apart from fulfilling the non-toxicity and structural requirements, synthetic materials are asked to direct cell response, offering engineered cues that provide specific instructions to cells. This work explores the functionalization of titanium with integrin-binding peptidomimetics as a novel and powerful strategy to improve the adhesion, proliferation and differentiation of osteoblast-like cells to implant materials. Such biomimetic strategy aims at targeting integrins αvβ3 and α5β1, which are highly expressed on osteoblasts and are essential for many fundamental functions in bone tissue development. The successful grafting of the bioactive molecules on titanium is proven by contact angle measurements, X-ray photoelectron spectroscopy and fluorescent labeling. Early attachment and spreading of cells are statistically enhanced by both peptidomimetics compared to unmodified titanium, reaching values of cell adhesion comparable to those obtained with full-length extracellular matrix proteins. Moreover, an increase in alkaline phosphatase activity, and statistically higher cell proliferation and mineralization are observed on surfaces coated with the peptidomimetics. This study shows an unprecedented biological activity for low-molecular-weight ligands on titanium, and gives striking evidence of the potential of these molecules to foster bone regeneration on implant materials.

  12. The RGD finger of Del-1 is a unique structural feature critical for integrin binding

    SciTech Connect

    Schürpf, Thomas; Chen, Qiang; Liu, Jin-huan; Wang, Rui; Springer, Timothy A.; Wang, Jia-huai

    2012-11-13

    Developmental endothelial cell locus-1 (Del-1) glycoprotein is secreted by endothelial cells and a subset of macrophages. Del-1 plays a regulatory role in vascular remodeling and functions in innate immunity through interaction with integrin {alpha}{sub V}{beta}{sub 3}. Del-1 contains 3 epidermal growth factor (EGF)-like repeats and 2 discoidin-like domains. An Arg-Gly-Asp (RGD) motif in the second EGF domain (EGF2) mediates adhesion by endothelial cells and phagocytes. We report the crystal structure of its 3 EGF domains. The RGD motif of EGF2 forms a type II' {beta} turn at the tip of a long protruding loop, dubbed the RGD finger. Whereas EGF2 and EGF3 constitute a rigid rod via an interdomain calcium ion binding site, the long linker between EGF1 and EGF2 lends considerable flexibility to EGF1. Two unique O-linked glycans and 1 N-linked glycan locate to the opposite side of EGF2 from the RGD motif. These structural features favor integrin binding of the RGD finger. Mutagenesis data confirm the importance of having the RGD motif at the tip of the RGD finger. A database search for EGF domain sequences shows that this RGD finger is likely an evolutionary insertion and unique to the EGF domain of Del-1 and its homologue milk fat globule-EGF 8. The RGD finger of Del-1 is a unique structural feature critical for integrin binding.

  13. The matricellular protein CCN5 regulates podosome function via interaction with integrin αvβ 3.

    PubMed

    Myers, Ronald B; Wei, Lan; Castellot, John J

    2014-06-01

    CCN proteins play crucial roles in cell motility, matrix turnover, and proliferation. In particular, CCN5 plays a role in cell motility and proliferation in several cell types; however, no functional binding proteins for CCN5 have been identified. In this study we report that CCN5 binds to the cell surface receptor integrin αvβ3 in vascular smooth muscle cells. Furthermore, this interaction takes place in podosomes, organelles known to degrade matrix and mediate motility. We show that CCN5 regulates the ability of podosomes to degrade matrix, but does not affect podosome formation. The level of CCN5 present in a podosome negatively correlates with its ability to degrade matrix. Conversely, knockdown of CCN5 greatly enhances the matrix-degrading ability of podosomes. These findings suggest that the antimotility effects of CCN5 may be mediated through the direct interaction of CCN5 and integrin αvβ3 in podosomes and the concomitant suppression of matrix degradation that is required for cell migration.

  14. The matricellular protein CCN1 suppresses lung cancer cell growth by inducing senescence via the p53/p21 pathway.

    PubMed

    Jim Leu, Shr-Jeng; Sung, Jung-Sung; Chen, Mei-Yu; Chen, Chih-Wei; Cheng, Jian-Yu; Wang, Tse-Yen; Wang, Jeng-Jung

    2013-09-01

    CCN1, a secreted matrix-associated molecule, is involved in multiple cellular processes. Previous studies have indicated that expression of CCN1 correlates inversely with the aggressiveness of non-small-cell lung carcinoma (NSCLC); however, the underlying mechanisms remain elusive. Using three NSCLC cell line systems, here we show that long-term treatment of cells with the recombinant CCN1 protein led to a permanent cell cycle arrest in G1 phase; cells remained viable as judged by apoptotic assays. CCN1-treated NSCLC cells acquired a phenotype characteristic of senescent cells, including an enlarged and flattened cell shape and expression of the senescence-associated β-galactosidase. Immunoblot analysis showed that addition of CCN1 increased the abundance of hypo-phosphorylated Rb, as well as accumulation of p53 and p21. Silencing the expression of p53 or p21 by lentivirus-mediated shRNA production in cells blocked the CCN1-induced senescence. Furthermore, a CCN1 mutant defective for binding integrin α6β1 and co-receptor heparan sulfate proteoglycans was incapable of senescence induction. Our finding that direct addition of CCN1 induces senescence in NSCLC cells provides a potential novel strategy for therapeutic intervention of lung cancers.

  15. Central corneal thickness does not correlate with TonoLab-measured IOP in several mouse strains with single transgenic mutations of matricellular proteins.

    PubMed

    Chatterjee, Ayan; Oh, Dong-Jin; Kang, Min Hyung; Rhee, Douglas J

    2013-10-01

    Accurate and reliable measurement of intraocular pressure (IOP) is crucial in the study of glaucoma using the mouse model. The purpose of this study was to determine the relationship between TonoLab-measured IOP and central corneal thickness (CCT) in mouse strains with single gene mutations of matricellular proteins. Wild-type (WT) and transgenic mouse strains with single gene mutations (KO) of thrombospondin-1 (TSP-1), thrombospondin-2 (TSP-2), osteopontin (OPN), hevin, and secreted protein acidic rich in cysteine (SPARC) were imaged at six weeks using optical coherence tomography (Stratus, Zeiss) to determine CCT. IOP was measured between 11am and 3pm using TonoLab, one week later. For all measurements, mice were anesthetized using intraperitoneal injection ketamine:xylazine. CCT and IOP were measured in 583 mice (TSP-1 n = 71 and 41, TSP-2 n = 60 and 32, OPN n = 81 and 50, hevin n = 59 and 76, SPARC n = 54 and 59, WT and KO, respectively). Mean CCT was 5-6% lower in three KO strains-TSP-1, OPN, and SPARC-compared to their corresponding WT (p = 1.55 × 10(-7), 1.63 × 10(-11), and 1.91 × 10(-7), respectively). The mean IOP was 8.3%, 6.6%, and 15.1% lower in three KO strains-TSP-1, TSP-2, and SPARC-compared to corresponding WT (p = 2.11 × 10(-5), 2.93 × 10(-3), and 3.76 × 10(-9), respectively. Linear regression of IOP versus CCT yielded no statistically significant within-strain correlations for TSP-1 (p = 0.12 and 0.073), TSP-2 (p = 0.473 and 0.92), OPN (p = 0.212 and 0.916), Hevin (p = 0.746 and 0.257), and SPARC (p = 0.080 and 0.056), reported as p-values considering a null hypothesis of zero slope (WT and KO, respectively). Neither C57-derived strains (TSP-1 and OPN) nor 129-derived strains (TSP-2, hevin, SPARC) demonstrated a correlation between mean IOP and mean CCT across different strains (p = 0.75 and p = 0.53, respectively). Taken together, these results indicate that CCT is not required to interpret Tono

  16. Neutrophil recruitment limited by high-affinity bent β2 integrin binding ligand in cis

    PubMed Central

    Fan, Zhichao; McArdle, Sara; Marki, Alex; Mikulski, Zbigniew; Gutierrez, Edgar; Engelhardt, Britta; Deutsch, Urban; Ginsberg, Mark; Groisman, Alex; Ley, Klaus

    2016-01-01

    Neutrophils are essential for innate immunity and inflammation and many neutrophil functions are β2 integrin-dependent. Integrins can extend (E+) and acquire a high-affinity conformation with an ‘open' headpiece (H+). The canonical switchblade model of integrin activation proposes that the E+ conformation precedes H+, and the two are believed to be structurally linked. Here we show, using high-resolution quantitative dynamic footprinting (qDF) microscopy combined with a homogenous conformation-reporter binding assay in a microfluidic device, that a substantial fraction of β2 integrins on human neutrophils acquire an unexpected E−H+ conformation. E−H+ β2 integrins bind intercellular adhesion molecules (ICAMs) in cis, which inhibits leukocyte adhesion in vitro and in vivo. This endogenous anti-inflammatory mechanism inhibits neutrophil aggregation, accumulation and inflammation. PMID:27578049

  17. Measurement of Cationic and Intracellular Modulation of Integrin Binding Affinity by AFM-Based Nanorobot

    PubMed Central

    Patterson, Kevin C.; Yang, Ruiguo; Zeng, Bixi; Song, Bo; Wang, Shouye; Xi, Ning; Basson, Marc D.

    2013-01-01

    Integrins are dynamic transmembrane cation-dependent heterodimers that both anchor cells in position and transduce signals into and out of cells. We used an atomic force microscope (AFM)-based nanorobotic system to measure integrin-binding forces in intact human intestinal epithelial Caco-2 cells. The AFM-based nanorobot enables human-directed, high-accuracy probe positioning and site-specific investigations. Functionalizing the AFM probe with an arginine-glycine-aspartate (RGD)-containing sequence (consensus binding sequence for integrins) allowed us to detect a series of peptide-cell membrane interactions with a median binding force of 115.1 ± 4.9 pN that were not detected in control interactions. Chelating divalent cations from the culture medium abolished these interactions, as did inhibiting intracellular focal adhesion kinase (FAK) using Y15. Adding 1 mM Mg2+ to the medium caused a rightward shift in the force-binding curve. Adding 1 mM Ca2+ virtually abolished the RGD-membrane specific interactions and blocked the Mg2+ effects. Cell adhesion assays demonstrated parallel effects of divalent cations and the FAK inhibitor on cell adhesion. These results demonstrate direct modulation of integrin-binding affinity by both divalent cations and intracellular signal inhibition. Additionally, three binding states (nonspecific, specific inactivated, and specific activated) were delineated from affinity measurements. Although other research has assumed that this process of integrin conformational change causes altered ligand binding, in this work we directly measured these three states in individual integrins in a physiologically based study. PMID:23823222

  18. Beta 1 integrin binding plays a role in the constant traction force generation in response to varying stiffness for cells grown on mature cardiac extracellular matrix.

    PubMed

    Gershlak, Joshua R; Black, Lauren D

    2015-01-15

    We have previously reported a unique response of traction force generation for cells grown on mature cardiac ECM, where traction force was constant over a range of stiffnesses. In this study we sought to further investigate the role of the complex mixture of ECM on this response and assess the potential mechanism behind it. Using traction force microscopy, we measured cellular traction forces and stresses for mesenchymal stem cells (MSCs) grown on polyacrylamide gels at a range of stiffnesses (9, 25, or 48 kPa) containing either adult rat heart ECM, different singular ECM proteins including collagen I, fibronectin, and laminin, or ECM mimics comprised of varying amounts of collagen I, fibronectin, and laminin. We also measured the expression of integrins on these different substrates as well as probed for β1 integrin binding. There was no significant change in traction force generation for cells grown on the adult ECM, as previously reported, whereas cells grown on singular ECM protein substrates had increased traction force generation with an increase in substrate stiffness. Cells grown on ECM mimics containing collagen I, fibronectin and laminin were found to be reminiscent of the traction forces generated by cells grown on native ECM. Integrin expression generally increased with increasing stiffness except for the β1 integrin, potentially implicating it as playing a role in the response to adult cardiac ECM. We inhibited binding through the β1 integrin on cells grown on the adult ECM and found that the inhibition of β1 binding led to a return to the typical response of increasing traction force generation with increasing stiffness. Our data demonstrates that cells grown on the mature cardiac ECM are able to circumvent typical stiffness related cellular behaviors, likely through β1 integrin binding to the complex composition.

  19. Matricellular signal transduction involving calmodulin in the social amoebozoan dictyostelium.

    PubMed

    O'Day, Danton H; Huber, Robert J

    2013-01-01

    The social amoebozoan Dictyostelium discoideum undergoes a developmental sequence wherein an extracellular matrix (ECM) sheath surrounds a group of differentiating cells. This sheath is comprised of proteins and carbohydrates, like the ECM of mammalian tissues. One of the characterized ECM proteins is the cysteine-rich, EGF-like (EGFL) repeat-containing, calmodulin (CaM)-binding protein (CaMBP) CyrA. The first EGFL repeat of CyrA increases the rate of random cell motility and cyclic AMP-mediated chemotaxis. Processing of full-length CyrA (~63 kDa) releases two major EGFL repeat-containing fragments (~45 kDa and ~40 kDa) in an event that is developmentally regulated. Evidence for an EGFL repeat receptor also exists and downstream intracellular signaling pathways involving CaM, Ras, protein kinase A and vinculin B phosphorylation have been characterized. In total, these results identify CyrA as a true matricellular protein comparable in function to tenascin C and other matricellular proteins from mammalian cells. Insight into the regulation and processing of CyrA has also been revealed. CyrA is the first identified extracellular CaMBP in this eukaryotic microbe. In keeping with this, extracellular CaM (extCaM) has been shown to be present in the ECM sheath where it binds to CyrA and inhibits its cleavage to release the 45 kDa and 40 kDa EGFL repeat-containing fragments. The presence of extCaM and its role in regulating a matricellular protein during morphogenesis extends our understanding of CaM-mediated signal transduction in eukaryotes. PMID:24705101

  20. Matricellular signal transduction involving calmodulin in the social amoebozoan dictyostelium.

    PubMed

    O'Day, Danton H; Huber, Robert J

    2013-02-15

    The social amoebozoan Dictyostelium discoideum undergoes a developmental sequence wherein an extracellular matrix (ECM) sheath surrounds a group of differentiating cells. This sheath is comprised of proteins and carbohydrates, like the ECM of mammalian tissues. One of the characterized ECM proteins is the cysteine-rich, EGF-like (EGFL) repeat-containing, calmodulin (CaM)-binding protein (CaMBP) CyrA. The first EGFL repeat of CyrA increases the rate of random cell motility and cyclic AMP-mediated chemotaxis. Processing of full-length CyrA (~63 kDa) releases two major EGFL repeat-containing fragments (~45 kDa and ~40 kDa) in an event that is developmentally regulated. Evidence for an EGFL repeat receptor also exists and downstream intracellular signaling pathways involving CaM, Ras, protein kinase A and vinculin B phosphorylation have been characterized. In total, these results identify CyrA as a true matricellular protein comparable in function to tenascin C and other matricellular proteins from mammalian cells. Insight into the regulation and processing of CyrA has also been revealed. CyrA is the first identified extracellular CaMBP in this eukaryotic microbe. In keeping with this, extracellular CaM (extCaM) has been shown to be present in the ECM sheath where it binds to CyrA and inhibits its cleavage to release the 45 kDa and 40 kDa EGFL repeat-containing fragments. The presence of extCaM and its role in regulating a matricellular protein during morphogenesis extends our understanding of CaM-mediated signal transduction in eukaryotes.

  1. Role of Heparan Sulfate in Cellular Infection of Integrin-Binding Coxsackievirus A9 and Human Parechovirus 1 Isolates.

    PubMed

    Merilahti, Pirjo; Karelehto, Eveliina; Susi, Petri

    2016-01-01

    Heparan sulfate/heparin class of proteoglycans (HSPG) have been shown to function in cellular attachment and infection of numerous viruses including picornaviruses. Coxsackievirus A9 (CV-A9) and human parechovirus 1 (HPeV-1) are integrin-binding members in the family Picornaviridae. CV-A9 Griggs and HPeV-1 Harris (prototype) strains have been reported not to bind to heparin, but it was recently shown that some CV-A9 isolates interact with heparin in vitro via VP1 protein with a specific T132R/K mutation. We found that the infectivity of both CV-A9 Griggs and HPeV-1 Harris was reduced by sodium chlorate and heparinase suggestive of HSPG interactions. We analyzed the T132 site in fifty-four (54) CV-A9 clinical isolates and found that only one of them possessed T132/R mutation while the other nine (9) had T132K. We then treated CV-A9 Griggs and HPeV-1 Harris and eight CV-A9 and six HPeV-1 clinical isolates with heparin and protamine. Although infectivity of Griggs strain was slightly reduced (by 25%), heparin treatment did not affect the infectivity of the CV-A9 isolates that do not possess the T132R/K mutation, which is in line with the previous findings. Some of the HPeV-1 isolates were also affected by heparin treatment, which suggested that there may be a specific heparin binding site in HPeV-1. In contrast, protamine (a specific inhibitor of heparin) completely inhibited the infection of both prototypes and clinical CV-A9 and HPeV-1 isolates. We conclude that T132R/K mutation has a role in heparin binding of CV-A9, but we also show data, which suggest that there are other HSPG binding sites in CV-A9. In all, we suggest that HSPGs play a general role in both CV-A9 and HPeV-1 infections.

  2. Role of Heparan Sulfate in Cellular Infection of Integrin-Binding Coxsackievirus A9 and Human Parechovirus 1 Isolates

    PubMed Central

    Merilahti, Pirjo; Karelehto, Eveliina; Susi, Petri

    2016-01-01

    Heparan sulfate/heparin class of proteoglycans (HSPG) have been shown to function in cellular attachment and infection of numerous viruses including picornaviruses. Coxsackievirus A9 (CV-A9) and human parechovirus 1 (HPeV-1) are integrin-binding members in the family Picornaviridae. CV-A9 Griggs and HPeV-1 Harris (prototype) strains have been reported not to bind to heparin, but it was recently shown that some CV-A9 isolates interact with heparin in vitro via VP1 protein with a specific T132R/K mutation. We found that the infectivity of both CV-A9 Griggs and HPeV-1 Harris was reduced by sodium chlorate and heparinase suggestive of HSPG interactions. We analyzed the T132 site in fifty-four (54) CV-A9 clinical isolates and found that only one of them possessed T132/R mutation while the other nine (9) had T132K. We then treated CV-A9 Griggs and HPeV-1 Harris and eight CV-A9 and six HPeV-1 clinical isolates with heparin and protamine. Although infectivity of Griggs strain was slightly reduced (by 25%), heparin treatment did not affect the infectivity of the CV-A9 isolates that do not possess the T132R/K mutation, which is in line with the previous findings. Some of the HPeV-1 isolates were also affected by heparin treatment, which suggested that there may be a specific heparin binding site in HPeV-1. In contrast, protamine (a specific inhibitor of heparin) completely inhibited the infection of both prototypes and clinical CV-A9 and HPeV-1 isolates. We conclude that T132R/K mutation has a role in heparin binding of CV-A9, but we also show data, which suggest that there are other HSPG binding sites in CV-A9. In all, we suggest that HSPGs play a general role in both CV-A9 and HPeV-1 infections. PMID:26785353

  3. Genotype tunes pancreatic ductal adenocarcinoma tissue tension to induce matricellular-fibrosis and tumor progression

    PubMed Central

    Laklai, Hanane; Miroshnikova, Yekaterina A.; Pickup, Michael W.; Collisson, Eric A.; Kim, Grace E.; Barrett, Alex S.; Hill, Ryan C.; Lakins, Johnathon N.; Schlaepfer, David D.; Mouw, Janna K.; LeBleu, Valerie S.; Roy, Nilotpal; Novitskiy, Sergey V.; Johansen, Julia S.; Poli, Valeria; Kalluri, Raghu; Iacobuzio-Donahue, Christine A.; Wood, Laura D.; Hebrok, Matthias; Hansen, Kirk; Moses, Harold L.; Weaver, Valerie M.

    2016-01-01

    Fibrosis compromises pancreatic ductal carcinoma (PDAC) treatment and contributes to patient mortality yet anti-stromal therapies are controversial. We found that human PDACs with impaired epithelial transforming growth factor β (TGF-β) signaling have elevated epithelial Stat3 activity and develop a stiffer, matricellular-enriched fibrosis associated with high epithelial tension and shorter patient survival. In several Kras-driven mouse models, both the loss of TGF-β signaling and elevated β1-integrin mechanosignaling engaged a positive feedback loop whereby Stat3 signaling promotes tumor progression by increasing matricellular fibrosis and tissue tension. In contrast, epithelial Stat3 ablation attenuated tumor progression by reducing the stromal stiffening and epithelial contractility induced by loss of TGF-β signaling. In PDAC patient biopsies, higher matricellular protein and activated Stat3 associated with SMAD4 mutation and shorter survival. The findings implicate epithelial tension and matricellular fibrosis in the aggressiveness of SMAD4 mutant pancreatic tumors, and highlight Stat3 and mechanics as key drivers of this phenotype. PMID:27089513

  4. Genotype tunes pancreatic ductal adenocarcinoma tissue tension to induce matricellular fibrosis and tumor progression.

    PubMed

    Laklai, Hanane; Miroshnikova, Yekaterina A; Pickup, Michael W; Collisson, Eric A; Kim, Grace E; Barrett, Alex S; Hill, Ryan C; Lakins, Johnathon N; Schlaepfer, David D; Mouw, Janna K; LeBleu, Valerie S; Roy, Nilotpal; Novitskiy, Sergey V; Johansen, Julia S; Poli, Valeria; Kalluri, Raghu; Iacobuzio-Donahue, Christine A; Wood, Laura D; Hebrok, Matthias; Hansen, Kirk; Moses, Harold L; Weaver, Valerie M

    2016-05-01

    Fibrosis compromises pancreatic ductal carcinoma (PDAC) treatment and contributes to patient mortality, yet antistromal therapies are controversial. We found that human PDACs with impaired epithelial transforming growth factor-β (TGF-β) signaling have high epithelial STAT3 activity and develop stiff, matricellular-enriched fibrosis associated with high epithelial tension and shorter patient survival. In several KRAS-driven mouse models, both the loss of TGF-β signaling and elevated β1-integrin mechanosignaling engaged a positive feedback loop whereby STAT3 signaling promotes tumor progression by increasing matricellular fibrosis and tissue tension. In contrast, epithelial STAT3 ablation attenuated tumor progression by reducing the stromal stiffening and epithelial contractility induced by loss of TGF-β signaling. In PDAC patient biopsies, higher matricellular protein and activated STAT3 were associated with SMAD4 mutation and shorter survival. The findings implicate epithelial tension and matricellular fibrosis in the aggressiveness of SMAD4 mutant pancreatic tumors and highlight STAT3 and mechanics as key drivers of this phenotype.

  5. ATP release due to Thy-1–integrin binding induces P2X7-mediated calcium entry required for focal adhesion formation

    PubMed Central

    Henríquez, Mauricio; Herrera-Molina, Rodrigo; Valdivia, Alejandra; Alvarez, Alvaro; Kong, Milene; Muñoz, Nicolás; Eisner, Verónica; Jaimovich, Enrique; Schneider, Pascal; Quest, Andrew F. G.; Leyton, Lisette

    2011-01-01

    Thy-1, an abundant mammalian glycoprotein, interacts with αvβ3 integrin and syndecan-4 in astrocytes and thus triggers signaling events that involve RhoA and its effector p160ROCK, thereby increasing astrocyte adhesion to the extracellular matrix. The signaling cascade includes calcium-dependent activation of protein kinase Cα upstream of Rho; however, what causes the intracellular calcium transients required to promote adhesion remains unclear. Purinergic P2X7 receptors are important for astrocyte function and form large non-selective cation pores upon binding to their ligand, ATP. Thus, we evaluated whether the intracellular calcium required for Thy-1-induced cell adhesion stems from influx mediated by ATP-activated P2X7 receptors. Results show that adhesion induced by the fusion protein Thy-1-Fc was preceded by both ATP release and sustained intracellular calcium elevation. Elimination of extracellular ATP with Apyrase, chelation of extracellular calcium with EGTA, or inhibition of P2X7 with oxidized ATP, all individually blocked intracellular calcium increase and Thy-1-stimulated adhesion. Moreover, Thy-1 mutated in the integrin-binding site did not trigger ATP release, and silencing of P2X7 with specific siRNA blocked Thy-1-induced adhesion. This study is the first to demonstrate a functional link between αvβ3 integrin and P2X7 receptors, and to reveal an important, hitherto unanticipated, role for P2X7 in calcium-dependent signaling required for Thy-1-stimulated astrocyte adhesion. PMID:21502139

  6. R-spondins: novel matricellular regulators of the skeleton.

    PubMed

    Knight, M Noelle; Hankenson, Kurt D

    2014-07-01

    R-spondins are a family of four matricellular proteins produced by a variety of cell-types. Structurally, R-spondins contain a TSR1 domain that retains the tryptophan structure and a modified cysteine-rich CSVCTG region. In addition, the R-spondins contain two furin repeats implicated in canonical Wnt signaling. R-spondins positively regulate canonical Wnt signaling by reducing Wnt receptor turnover and thereby increasing beta-catenin stabilization. R-spondins are prominently expressed in the developing skeleton and contribute to limb formation, particularly of the distal digit. Additionally, results suggest that R-spondins may contribute to the maintenance of adult bone mass by regulating osteoblastogenesis and bone formation.

  7. Tumor suppressor Smad4 mediates downregulation of the anti-adhesive invasion-promoting matricellular protein SPARC: Landscaping activity of Smad4 as revealed by a "secretome" analysis.

    PubMed

    Volmer, Martin W; Radacz, Yvonne; Hahn, Stephan A; Klein-Scory, Susanne; Stühler, Kai; Zapatka, Marc; Schmiegel, Wolff; Meyer, Helmut E; Schwarte-Waldhoff, Irmgard

    2004-05-01

    We have demonstrated previously that restoration of Smad4 expression in Smad4-deficient SW480 human colon carcinoma cells was adequate to suppress tumorigenicity and invasive potential, whereas cell growth in vitro was not affected. Here we show that Smad4-positive and Smad4-negative SW480 cells deposit extracellular matrices in tissue culture which are functionally different with respect to their adhesiveness. We present a "differential secretomics analysis" as the most direct approach to identify the underlying alterations. The protein composition of conditioned media produced by Smad4-positive and Smad4-negative SW480 cells was compared by two-dimensional (2-D) gel electrophoresis. A major group of protein spots was detected in media derived from Smad4-negative cells, only, which were identified as "secreted protein, acidic and rich in cysteins" (SPARC) by mass spectrometry. SPARC expression in SW480 cells was suppressed by Smad4 at the level of transcription. SPARC is a glycoprotein of the extracellular matrix, characterized as an anti-adhesive and invasion-promoting protein. Differential secretomics appeared as a powerful method to identify a novel Smad4 target gene, which may be one of the players involved in reduced adhesiveness of extracellular matrices and thus consistent with Smad4's emerging role as an invasion suppressor.

  8. Identification of a novel integrin alphaMbeta2 binding site in CCN1 (CYR61), a matricellular protein expressed in healing wounds and atherosclerotic lesions.

    PubMed

    Schober, Joseph M; Lau, Lester F; Ugarova, Tatiana P; Lam, Stephen C-T

    2003-07-11

    CCN1 (cysteine-rich 61) and CCN2 (connective tissue growth factor) are growth factor-inducible immediate-early gene products found in atherosclerotic lesions, restenosed blood vessels, and healing cutaneous wounds. Both CCN proteins have been shown to support cell adhesion and induce cell migration through interaction with integrin receptors. Recently, we have identified integrin alphaMbeta2 as the major adhesion receptor mediating monocyte adhesion to CCN1 and CCN2 and have shown that the alphaMI domain binds specifically to both proteins. In the present study, we demonstrated that activated monocytes adhered to a synthetic peptide (CCN1-H2, SSVKKYRPKYCGS) derived from a conserved region within the CCN1 C-terminal domain, and this process was blocked by the anti-alphaM monoclonal antibody 2LPM19c. Consistently, a glutathione S-transferase (GST) fusion protein containing the alphaMI domain (GST-alphaMI) bound to immobilized CCN1-H2 as well as to the corresponding H2 sequence in CCN2 (CCN2-H2, TSVKTYRAKFCGV). By contrast, a scrambled CCN1-H2 peptide and an 18-residue peptide derived from an adjacent sequence of CCN1-H2 failed to support monocyte adhesion or alphaMI domain binding. To confirm that the CCN1-H2 sequence within the CCN1 protein mediates alphaMbeta2 interaction, we developed an anti-peptide antibody against CCN1-H2 and showed that it specifically blocked GST-alphaMI binding to intact CCN1. Collectively, these results identify the H2 sequence in CCN1 and CCN2 as a novel integrin alphaMbeta2 binding motif that bears no apparent homology to any alphaMbeta2 binding sequence reported to date.

  9. Attenuation of increased secretory leukocyte protease inhibitor, matricellular proteins and angiotensin II and left ventricular remodeling by candesartan and omapatrilat during healing after reperfused myocardial infarction.

    PubMed

    Palaniyappan, Ariv; Uwiera, Richard R E; Idikio, Halliday; Menon, Vijay; Jugdutt, Catherine; Jugdutt, Bodh I

    2013-04-01

    While secretory-leukocyte-protease-inhibitor (SLPI) may promote skin wound healing, its role in infarct healing after reperfused myocardial infarction (RMI) remains unclear. Short-term intravenous angiotensin II (AngII) receptor blocker therapy with candesartan (CN) attenuates increased SLPI and markers of early matrix/left ventricular (LV) in acute RMI. To determine whether reducing effects of AngII with CN or the vasopeptidase inhibitor omapatrilat (OMA) during the healing phase after RMI attenuates SLPI and other mediators of healing and matrix/LV remodeling, we measured these in Sprague-Dawley rats randomized to oral placebo, CN (30 mg/kg/day) or OMA (10 mg/kg/day) therapy during healing between days 2 and 23 after RMI and sham. On day-25, RMI-placebo showed significant LV remodeling, systolic/diastolic dysfunction and impaired passive compliance, and ischemic zone increases in SLPI, secreted-protein-acidic-and-rich-in-cysteine (SPARC) and osteopontin (OPN) mRNA and protein. In addition, metalloproteinase (MMP)-9 and -2, a-disintegrin-and-metalloproteinase (ADAM)-10 and -17, inducible-nitric-oxide-synthase (iNOS), pro-inflammatory cytokines interleukin (IL)-6, and tumor necrosis factor-α, transforming growth factor (TGF)-β(1) and its signaling molecule p-Smad-2, myeloperoxidase (MPO), AngII, MPO-positive granulocytes, MAC387-positive macrophages and monocytes, scar collagens, cardiomyocyte and fibroblast apoptosis, and microvascular no-reflow also increased whereas anti-inflammatory cytokine IL-10 decreased. Both CN and OMA attenuated all the changes except IL-10, which normalized. Thus, CN or OMA treatment during healing after RMI results in attenuation of SLPI as well as tissue AngII and mediators of inflammation and matrix/LV remodeling including SPARC, OPN, and ADAMs. Whether increasing SLPI on top of background AngII inhibition or therapy such as CN or OMA might produce added remodeling benefit needs study.

  10. The matricellular functions of small leucine-rich proteoglycans (SLRPs).

    PubMed

    Merline, Rosetta; Schaefer, Roland M; Schaefer, Liliana

    2009-12-01

    The small leucine-rich proteoglycans (SLRPs) are biologically active components of the extracellular matrix (ECM), consisting of a protein core with leucine rich-repeat (LRR) motifs covalently linked to glycosaminoglycan (GAG) side chains. The diversity in composition resulting from the various combinations of protein cores substituted with one or more GAG chains along with their pericellular localization enables SLRPs to interact with a host of different cell surface receptors, cytokines, growth factors, and other ECM components, leading to modulation of cellular functions. SLRPs are capable of binding to: (i) different types of collagens, thereby regulating fibril assembly, organization, and degradation; (ii) Toll-like receptors (TLRs), complement C1q, and tumor necrosis factor-alpha (TNFalpha), regulating innate immunity and inflammation; (iii) epidermal growth factor receptor (EGF-R), insulin-like growth factor receptor (IGF-IR), and c-Met, influencing cellular proliferation, survival, adhesion, migration, tumor growth and metastasis as well as synthesis of other ECM components; (iv) low-density lipoprotein receptor-related protein (LRP-1) and TGF-beta, modulating cytokine activity and fibrogenesis; and (v) growth factors such as bone morphogenic protein (BMP-4) and Wnt-I-induced secreted protein-1 (WISP-1), controlling cell proliferation and differentiation. Thus, the ability of SLRPs, as ECM components, to directly or indirectly regulate cell-matrix crosstalk, resulting in the modulation of various biological processes, aptly qualifies these compounds as matricellular proteins.

  11. Two RGD-independent alpha vbeta 3 integrin binding sites on tumstatin regulate distinct anti-tumor properties.

    PubMed

    Maeshima, Y; Colorado, P C; Kalluri, R

    2000-08-01

    Vascular basement membrane is an important regulator of angiogenesis and undergoes many alterations during angiogenesis and these changes are speculated to influence neovascularization. Recently, fragments of collagen molecules have been identified to possess anti-angiogenic activity. Tumstatin (alpha3(IV)NC1 domain) is one such novel molecule with distinct anti-tumor properties and possesses an N-terminal (amino acids 54-132) anti-angiogenic and a C-terminal (amino acids 185-203) anti-tumor cell activity (Maeshima, Y., et al. 2000) J. Biol. Chem. 275, 21340-21348). Previous studies have identified the 185-203 amino acid sequence as a ligand for alpha(v)beta(3) integrin (Shahan, T. A., et al. (1999) Cancer Res. 59, 4584-4590). In the present study, we found distinct additional RGD-independent alpha(v)beta(3) integrin binding site within 54-132 amino acids of tumstatin. This site is not essential for inhibition of tumor cell proliferation but necessary for the anti-angiogenic activity. A fragment of tumstatin containing 54-132 amino acid (tum-2) binds both endothelial cells and melanoma cells but only inhibited proliferation of endothelial cells, with no effect on tumor cell proliferation. A similar experiment with fragment of tumstatin containing the 185-203 amino acid (tum-4) demonstrates that it binds both endothelial cells and melanoma cells but only inhibits the proliferation of melanoma cells. The presence of cyclic RGD peptides did not affect the alpha(v)beta(3) integrin-mediated activity of tumstatin, although significant inhibition of endothelial cell binding to vitronectin was observed. The two distinct RGD-independent binding sites on tumstatin suggest unique alpha(v)beta(3) integrin-mediated mechanisms governing the two distinct anti-tumor properties of tumstatin. PMID:10837460

  12. Conformation and concerted dynamics of the integrin-binding site and the C-terminal region of echistatin revealed by homonuclear NMR

    PubMed Central

    Monleón, Daniel; Esteve, Vicent; Kovacs, Helena; Calvete, Juan J.; Celda, Bernardo

    2004-01-01

    Echistatin is a potent antagonist of the integrins αvβ3, α5β1 and αIIbβ3. Its full inhibitory activity depends on an RGD (Arg-Gly-Asp) motif expressed at the tip of the integrin-binding loop and on its C-terminal tail. Previous NMR structures of echistatin showed a poorly defined integrin-recognition sequence and an incomplete C-terminal tail, which left the molecular basis of the functional synergy between the RGD loop and the C-terminal region unresolved. We report a high-resolution structure of echistatin and an analysis of its internal motions by off-resonance ROESY (rotating-frame Overhauser enhancement spectroscopy). The full-length C-terminal polypeptide is visible as a β-hairpin running parallel to the RGD loop and exposing at the tip residues Pro43, His44 and Lys45. The side chains of the amino acids of the RGD motif have well-defined conformations. The integrin-binding loop displays an overall movement with maximal amplitude of 30°. Internal angular motions in the 100–300 ps timescale indicate increased flexibility for the backbone atoms at the base of the integrin-recognition loop. In addition, backbone atoms of the amino acids Ala23 (flanking the R24GD26 tripeptide) and Asp26 of the integrin-binding motif showed increased angular mobility, suggesting the existence of major and minor hinge effects at the base and the tip, respectively, of the RGD loop. A strong network of NOEs (nuclear Overhauser effects) between residues of the RGD loop and the C-terminal tail indicate concerted motions between these two functional regions. A full-length echistatin–αvβ3 docking model suggests that echistatin's C-terminal amino acids may contact αv-subunit residues and provides new insights to delineate structure–function correlations. PMID:15535803

  13. Lymphocyte function-associated antigen-1 binding residues in intercellular adhesion molecule-2 (ICAM-2) and the integrin binding surface in the ICAM subfamily

    PubMed Central

    Casasnovas, José M.; Pieroni, Cristiana; Springer, Timothy A.

    1999-01-01

    The crystal structure of intercellular adhesion molecule-2 (ICAM-2) revealed significant differences in the presentation of the critical acidic residue important for integrin binding between I and non-I-domain integrin ligands. Based on this crystal structure, we mutagenized ICAM-2 to localize the binding site for the integrin lymphocyte function-associated antigen-1 (LFA-1). The integrin binding site runs diagonally across the GFC β-sheet and includes residues on the CD edge of the β-sandwich. The site is oblong and runs along a flat ridge on the upper half of domain 1, which is proposed to dock to a groove in the I domain of LFA-1, with the critical Glu-37 residue ligating the Mg2+ in the I domain. Previous mutagenesis of ICAM-1 and ICAM-3, interpreted in light of the recently determined ICAM-1 and ICAM-2 structures, suggests similar binding sites. By contrast, major differences are seen with vascular cell adhesion molecule-1 (VCAM-1), which binds α4 integrins that lack an I domain. The binding site on VCAM-1 includes the lower portion of domain 1 and the upper part of domain 2, whereas the LFA-1 binding site on ICAM is confined to the upper part of domain 1. PMID:10077629

  14. Lymphocyte function-associated antigen-1 binding residues in intercellular adhesion molecule-2 (ICAM-2) and the integrin binding surface in the ICAM subfamily.

    PubMed

    Casasnovas, J M; Pieroni, C; Springer, T A

    1999-03-16

    The crystal structure of intercellular adhesion molecule-2 (ICAM-2) revealed significant differences in the presentation of the critical acidic residue important for integrin binding between I and non-I-domain integrin ligands. Based on this crystal structure, we mutagenized ICAM-2 to localize the binding site for the integrin lymphocyte function-associated antigen-1 (LFA-1). The integrin binding site runs diagonally across the GFC beta-sheet and includes residues on the CD edge of the beta-sandwich. The site is oblong and runs along a flat ridge on the upper half of domain 1, which is proposed to dock to a groove in the I domain of LFA-1, with the critical Glu-37 residue ligating the Mg2+ in the I domain. Previous mutagenesis of ICAM-1 and ICAM-3, interpreted in light of the recently determined ICAM-1 and ICAM-2 structures, suggests similar binding sites. By contrast, major differences are seen with vascular cell adhesion molecule-1 (VCAM-1), which binds alpha4 integrins that lack an I domain. The binding site on VCAM-1 includes the lower portion of domain 1 and the upper part of domain 2, whereas the LFA-1 binding site on ICAM is confined to the upper part of domain 1. PMID:10077629

  15. Screening of integrin-binding peptides in a laminin peptide library derived from the mouse laminin β chain short arm regions.

    PubMed

    Katagiri, Fumihiko; Takagi, Masaharu; Nakamura, Minako; Tanaka, Yoichiro; Hozumi, Kentaro; Kikkawa, Yamato; Nomizu, Motoyoshi

    2014-05-15

    Laminins, major components of basement membrane, consist of three different subunits, α, β, and γ chains, and so far, five α, three β, and three γ chains have been identified. We have constructed synthetic peptide libraries derived from the laminin sequences and identified various cell-adhesive peptides. Ten active peptides from the laminin α chain sequences (α1-α5) were found to promote integrin-mediated cell adhesion. Previously, we found fourteen cell-adhesive peptides from the β1 chain sequence but their receptors have not been analyzed. Here, we expanded the synthetic peptide library to add peptides from the short arm regions of the laminin β2 and β3 chains and screened for integrin-binding peptides. Twenty-seven peptides promoted human dermal fibroblast (HDF) attachment in a peptide-coated plate assay. The morphological appearance of HDFs on the peptide-coated plates differed depending on the peptides. B34 (REKYYYAVYDMV, mouse laminin β1 chain, 255-266), B67 (IPYSMEYEILIRY, mouse laminin β1 chain, 604-616), B2-105 (APNFWNFTSGRG, mouse laminin β2 chain, 1081-1092), and B3-19 (GHLTGGKVQLNL, mouse laminin β3 chain, 182-193) promoted HDF spreading and HDF attachment was inhibited by EDTA, suggesting that the peptides interact with integrins. Immunostaining analyses revealed that B67 induced well-organized actin stress fibers and focal contacts containing vinculin, however, B34, B2-105, and B3-19 did not exhibit stress fiber formation or focal contacts. The inhibition assay using anti-integrin antibodies indicated that B67 interacts with α3, α6, and β1 integrins, and B34 and B3-19 interact with β1 integrin. Based on adhesion analysis of peptides modified with an alanine scan and on switching analysis with the homologous inactive sequence B2-64 (LPRAMDYDLLLRW, mouse laminin β2 chain, 618-630), the Glu(8) residue in the B67 peptide was critical for HDF adhesion. These findings are useful for identifying an integrin binding motif. The B67 peptide

  16. Regulation of soluble guanylate cyclase by matricellular thrombospondins: implications for blood flow.

    PubMed

    Rogers, Natasha M; Seeger, Franziska; Garcin, Elsa D; Roberts, David D; Isenberg, Jeffrey S

    2014-01-01

    Nitric oxide (NO) maintains cardiovascular health by activating soluble guanylate cyclase (sGC) to increase cellular cGMP levels. Cardiovascular disease is characterized by decreased NO-sGC-cGMP signaling. Pharmacological activators and stimulators of sGC are being actively pursued as therapies for acute heart failure and pulmonary hypertension. Here we review molecular mechanisms that modulate sGC activity while emphasizing a novel biochemical pathway in which binding of the matricellular protein thrombospondin-1 (TSP1) to the cell surface receptor CD47 causes inhibition of sGC. We discuss the therapeutic implications of this pathway for blood flow, tissue perfusion, and cell survival under physiologic and disease conditions.

  17. ß1 Integrin Binding Phosphorylates Ezrin at T567 to Activate a Lipid Raft Signalsome Driving Invadopodia Activity and Invasion

    PubMed Central

    Antelmi, Ester; Cardone, Rosa A.; Greco, Maria R.; Rubino, Rosa; Di Sole, Francesca; Martino, Nicola A.; Casavola, Valeria; Carcangiu, MariaLuisa; Moro, Loredana; Reshkin, Stephan J.

    2013-01-01

    Extracellular matrix (ECM) degradation is a critical process in tumor cell invasion and requires matrix degrading protrusions called invadopodia. The Na+/H+ exchanger (NHE1) has recently been shown to be fundamental in the regulation of invadopodia actin cytoskeleton dynamics and activity. However, the structural link between the invadopodia cytoskeleton and NHE1 is still unknown. A candidate could be ezrin, a linker between the NHE1 and the actin cytoskeleton known to play a pivotal role in invasion and metastasis. However, the mechanistic basis for its role remains unknown. Here, we demonstrate that ezrin phosphorylated at T567 is highly overexpressed in the membrane of human breast tumors and positively associated with invasive growth and HER2 overexpression. Further, in the metastatic cell line, MDA-MB-231, p-ezrin was almost exclusively expressed in invadopodia lipid rafts where it co-localized in a functional complex with NHE1, EGFR, ß1-integrin and phosphorylated-NHERF1. Manipulation by mutation of ezrins T567 phosphorylation state and/or PIP2 binding capacity or of NHE1s binding to ezrin or PIP2 demonstrated that p-ezrin expression and binding to PIP2 are required for invadopodia-mediated ECM degradation and invasion and identified NHE1 as the membrane protein that p-ezrin regulates to induce invadopodia formation and activity. PMID:24086451

  18. Matricryptins Network with Matricellular Receptors at the Surface of Endothelial and Tumor Cells

    PubMed Central

    Ricard-Blum, Sylvie; Vallet, Sylvain D.

    2016-01-01

    The extracellular matrix (ECM) is a source of bioactive fragments called matricryptins or matrikines resulting from the proteolytic cleavage of extracellular proteins (e.g., collagens, elastin, and laminins) and proteoglycans (e.g., perlecan). Matrix metalloproteinases (MMPs), cathepsins, and bone-morphogenetic protein-1 release fragments, which regulate physiopathological processes including tumor growth, metastasis, and angiogenesis, a pre-requisite for tumor growth. A number of matricryptins, and/or synthetic peptides derived from them, are currently investigated as potential anti-cancer drugs both in vitro and in animal models. Modifications aiming at improving their efficiency and their delivery to their target cells are studied. However, their use as drugs is not straightforward. The biological activities of these fragments are mediated by several receptor families. Several matricryptins may bind to the same matricellular receptor, and a single matricryptin may bind to two different receptors belonging or not to the same family such as integrins and growth factor receptors. Furthermore, some matricryptins interact with each other, integrins and growth factor receptors crosstalk and a signaling pathway may be regulated by several matricryptins. This forms an intricate 3D interaction network at the surface of tumor and endothelial cells, which is tightly associated with other cell-surface associated molecules such as heparan sulfate, caveolin, and nucleolin. Deciphering the molecular mechanisms underlying the behavior of this network is required in order to optimize the development of matricryptins as anti-cancer agents. PMID:26869928

  19. Matricryptins Network with Matricellular Receptors at the Surface of Endothelial and Tumor Cells.

    PubMed

    Ricard-Blum, Sylvie; Vallet, Sylvain D

    2016-01-01

    The extracellular matrix (ECM) is a source of bioactive fragments called matricryptins or matrikines resulting from the proteolytic cleavage of extracellular proteins (e.g., collagens, elastin, and laminins) and proteoglycans (e.g., perlecan). Matrix metalloproteinases (MMPs), cathepsins, and bone-morphogenetic protein-1 release fragments, which regulate physiopathological processes including tumor growth, metastasis, and angiogenesis, a pre-requisite for tumor growth. A number of matricryptins, and/or synthetic peptides derived from them, are currently investigated as potential anti-cancer drugs both in vitro and in animal models. Modifications aiming at improving their efficiency and their delivery to their target cells are studied. However, their use as drugs is not straightforward. The biological activities of these fragments are mediated by several receptor families. Several matricryptins may bind to the same matricellular receptor, and a single matricryptin may bind to two different receptors belonging or not to the same family such as integrins and growth factor receptors. Furthermore, some matricryptins interact with each other, integrins and growth factor receptors crosstalk and a signaling pathway may be regulated by several matricryptins. This forms an intricate 3D interaction network at the surface of tumor and endothelial cells, which is tightly associated with other cell-surface associated molecules such as heparan sulfate, caveolin, and nucleolin. Deciphering the molecular mechanisms underlying the behavior of this network is required in order to optimize the development of matricryptins as anti-cancer agents.

  20. Matricellular protein periostin contributes to hepatic inflammation and fibrosis.

    PubMed

    Huang, Yangmei; Liu, Weiping; Xiao, Hongjun; Maitikabili, Alaiyi; Lin, Qinghua; Wu, Tiantian; Huang, Zhengjie; Liu, Fan; Luo, Qi; Ouyang, Gaoliang

    2015-03-01

    Periostin actively contributes to tissue injury, fibrosis, atherosclerosis, and inflammatory diseases; however, its role in hepatic fibrosis is unclear. Herein, we revealed that periostin expression was significantly up-regulated in carbon tetrachloride- and bile duct ligation-induced mice with acute and chronic liver fibrosis. Deficiency in periostin abrogated the development of liver fibrosis in mice. Carbon tetrachloride treatment significantly increased α-smooth muscle actin, fibronectin, and collagen I levels in wild-type mice, which were unaffected in periostin-knockout mice. Periostin-deficient mice showed a significantly reduced area of collagen deposition and decreased levels of serum alanine aminotransferase and aspartate aminotransferase compared with wild-type mice after 2 weeks of carbon tetrachloride administration. Chemokine ligand 2, IL-6, IL-1β, tumor necrosis factor-α, and tissue inhibitor of metalloproteinases 1 mRNA levels were significantly lower in periostin-deficient mice than in wild-type mice after carbon tetrachloride treatment. Periostin colocalized with hepatic stellate cell-derived collagen I and α-smooth muscle actin in mouse acute and chronic fibrotic liver tissues. Transforming growth factor (TGF)-β1 markedly induced periostin expression in primary mouse hepatic stellate cells. Periostin-deficient mice showed significantly lower levels of TGF-β1 and TGF-β2 compared with wild-type mice after carbon tetrachloride treatment. High levels of periostin in patients with acute or chronic hepatitis correlated with TGF-β1 and TGF-β2 expression in serum from patients with hepatitis. Data indicate that periostin is a novel mediator of hepatic fibrosis development.

  1. The Matricellular Receptor LRP1 Forms an Interface for Signaling and Endocytosis in Modulation of the Extracellular Tumor Environment.

    PubMed

    Van Gool, Bart; Dedieu, Stéphane; Emonard, Hervé; Roebroek, Anton J M

    2015-01-01

    The membrane protein low-density lipoprotein receptor related-protein 1 (LRP1) has been attributed a role in cancer. However, its presumably often indirect involvement is far from understood. LRP1 has both endocytic and signaling activities. As a matricellular receptor it is involved in regulation, mostly by clearing, of various extracellular matrix degrading enzymes including matrix metalloproteinases, serine proteases, protease inhibitor complexes, and the endoglycosidase heparanase. Furthermore, by binding extracellular ligands including growth factors and subsequent intracellular interaction with scaffolding and adaptor proteins it is involved in regulation of various signaling cascades. LRP1 expression levels are often downregulated in cancer and some studies consider low LRP1 levels a poor prognostic factor. On the contrary, upregulation in brain cancers has been noted and clinical trials explore the use of LRP1 as cargo receptor to deliver cytotoxic agents. This mini-review focuses on LRP1's role in tumor growth and metastasis especially by modulation of the extracellular tumor environment. In relation to this role its diagnostic, prognostic and therapeutic potential will be discussed. PMID:26617523

  2. The Matricellular Receptor LRP1 Forms an Interface for Signaling and Endocytosis in Modulation of the Extracellular Tumor Environment

    PubMed Central

    Van Gool, Bart; Dedieu, Stéphane; Emonard, Hervé; Roebroek, Anton J. M.

    2015-01-01

    The membrane protein low-density lipoprotein receptor related-protein 1 (LRP1) has been attributed a role in cancer. However, its presumably often indirect involvement is far from understood. LRP1 has both endocytic and signaling activities. As a matricellular receptor it is involved in regulation, mostly by clearing, of various extracellular matrix degrading enzymes including matrix metalloproteinases, serine proteases, protease inhibitor complexes, and the endoglycosidase heparanase. Furthermore, by binding extracellular ligands including growth factors and subsequent intracellular interaction with scaffolding and adaptor proteins it is involved in regulation of various signaling cascades. LRP1 expression levels are often downregulated in cancer and some studies consider low LRP1 levels a poor prognostic factor. On the contrary, upregulation in brain cancers has been noted and clinical trials explore the use of LRP1 as cargo receptor to deliver cytotoxic agents. This mini-review focuses on LRP1’s role in tumor growth and metastasis especially by modulation of the extracellular tumor environment. In relation to this role its diagnostic, prognostic and therapeutic potential will be discussed. PMID:26617523

  3. The Matricellular Receptor LRP1 Forms an Interface for Signaling and Endocytosis in Modulation of the Extracellular Tumor Environment.

    PubMed

    Van Gool, Bart; Dedieu, Stéphane; Emonard, Hervé; Roebroek, Anton J M

    2015-01-01

    The membrane protein low-density lipoprotein receptor related-protein 1 (LRP1) has been attributed a role in cancer. However, its presumably often indirect involvement is far from understood. LRP1 has both endocytic and signaling activities. As a matricellular receptor it is involved in regulation, mostly by clearing, of various extracellular matrix degrading enzymes including matrix metalloproteinases, serine proteases, protease inhibitor complexes, and the endoglycosidase heparanase. Furthermore, by binding extracellular ligands including growth factors and subsequent intracellular interaction with scaffolding and adaptor proteins it is involved in regulation of various signaling cascades. LRP1 expression levels are often downregulated in cancer and some studies consider low LRP1 levels a poor prognostic factor. On the contrary, upregulation in brain cancers has been noted and clinical trials explore the use of LRP1 as cargo receptor to deliver cytotoxic agents. This mini-review focuses on LRP1's role in tumor growth and metastasis especially by modulation of the extracellular tumor environment. In relation to this role its diagnostic, prognostic and therapeutic potential will be discussed.

  4. Venom gland EST analysis of the saw-scaled viper, Echis ocellatus, reveals novel alpha9beta1 integrin-binding motifs in venom metalloproteinases and a new group of putative toxins, renin-like aspartic proteases.

    PubMed

    Wagstaff, Simon C; Harrison, Robert A

    2006-08-01

    Echis ocellatus is the most medically important snake in West Africa. However, the composition of its venom and the differential contribution of these venom components to the severe haemorrhagic and coagulopathic pathology of envenoming are poorly understood. To address this situation we assembled a toxin transcriptome based upon 1000 expressed sequence tags (EST) from a cDNA library constructed from pooled venom glands of 10 individual E. ocellatus. We used a variety of bioinformatic tools to construct a fully annotated venom-toxin transcriptome that was interrogated with a combination of BLAST annotation, gene ontology cataloguing and disintegrin-motif searching. The results of these analyses revealed an unusually abundant and diverse expression of snake venom metalloproteinases (SVMP) and a broad toxin-expression profile including several distinct isoforms of bradykinin-potentiating peptides, phospholipase A(2), C-type lectins, serine proteinases and l-amino oxidases. Most significantly, we identified for the first time a conserved alpha(9)beta(1) integrin-binding motif in several SVMPs, and a new group of putative venom toxins, renin-like aspartic proteases. PMID:16713134

  5. Matrikine and matricellular regulators of EGF receptor signaling on cancer cell migration and invasion.

    PubMed

    Grahovac, Jelena; Wells, Alan

    2014-01-01

    Cancer invasion is a complex process requiring, among other events, extensive remodeling of the extracellular matrix including deposition of pro-migratory and pro-proliferative moieties. In recent years, it has been described that while invading through matrices cancer cells can change shape and adapt their migration strategies depending on the microenvironmental context. Although intracellular signaling pathways governing the mesenchymal to amoeboid migration shift and vice versa have been mostly elucidated, the extracellular signals promoting these shifts are largely unknown. In this review, we summarize findings that point to matrikines that bind specifically to the EGF receptor as matricellular molecules that enable cancer cell migrational plasticity and promote invasion.

  6. Molecular dynamics and docking simulation of a natural variant of Activated Protein C with impaired protease activity: implications for integrin-mediated antiseptic function.

    PubMed

    D'Ursi, Pasqualina; Orro, Alessandro; Morra, Giulia; Moscatelli, Marco; Trombetti, Gabriele; Milanesi, Luciano; Rovida, Ermanna

    2015-01-01

    Activated Protein C (APC) is a multifunctional serine protease, primarily known for its anticoagulant function in the coagulation system. Several studies have already elucidated its role in counteracting apoptosis and inflammation in cells, while significant effort is still ongoing for defining its involvement in sepsis. Earlier literature has shown that the antiseptic function of APC is mediated by its binding to leukocyte integrins, which is due to the presence of the integrin binding motif Arg-Gly-Asp at the N-terminus of the APC catalytic chain. Many natural mutants have been identified in patients with Protein C deficiency diagnosis including a variant of specificity pocket (Gly216Asp). In this work, we present a molecular model of the complex of APC with αVβ3 integrin obtained by protein-protein docking approach. A computational analysis of this variant is hereby presented, based on molecular dynamics and docking simulations, aiming at investigating the effects of the Gly216Asp mutation on the protein conformation and inferring its functional implications. Our study shows that such mutation is likely to impair the protease activity while preserving the overall protein fold. Moreover, superposition of the integrin binding motifs in wild-type and mutant forms suggests that the interaction with integrin can still occur and thus the mutant is likely to retain its antiseptic function related to the neutrophyl integrin binding. Therapeutic applications could result in this APC mutant which retains antiseptic function without anticoagulant side effects.

  7. Cyr61, a matricellular protein, is needed for dendritic arborization of hippocampal neurons.

    PubMed

    Malik, Anna R; Urbanska, Malgorzata; Gozdz, Agata; Swiech, Lukasz J; Nagalski, Andrzej; Perycz, Malgorzata; Blazejczyk, Magdalena; Jaworski, Jacek

    2013-03-22

    The shape of the dendritic arbor is one of the criteria of neuron classification and reflects functional specialization of particular classes of neurons. The development of a proper dendritic branching pattern strongly relies on interactions between the extracellular environment and intracellular processes responsible for dendrite growth and stability. We previously showed that mammalian target of rapamycin (mTOR) kinase is crucial for this process. In this work, we performed a screen for modifiers of dendritic growth in hippocampal neurons, the expression of which is potentially regulated by mTOR. As a result, we identified Cyr61, an angiogenic factor with unknown neuronal function, as a novel regulator of dendritic growth, which controls dendritic growth in a β1-integrin-dependent manner.

  8. The matricellular protein CCN3 regulates NOTCH1 signalling in chronic myeloid leukaemia.

    PubMed

    Suresh, Sukanya; McCallum, Lynn; Crawford, Lisa J; Lu, Wan Hua; Sharpe, Daniel J; Irvine, Alexandra E

    2013-11-01

    Deregulated NOTCH1 has been reported in lymphoid leukaemia, although its role in chronic myeloid leukaemia (CML) is not well established. We previously reported BCR-ABL down-regulation of a novel haematopoietic regulator, CCN3, in CML; CCN3 is a non-canonical NOTCH1 ligand. This study characterizes the NOTCH1–CCN3 signalling axis in CML. In K562 cells, BCR-ABL silencing reduced full-length NOTCH1 (NOTCH1-FL) and inhibited the cleavage of NOTCH1 intracellular domain (NOTCH1-ICD), resulting in decreased expression of the NOTCH1 targets c-MYC and HES1. K562 cells stably overexpressing CCN3 (K562/CCN3) or treated with recombinant CCN3(rCCN3) showed a significant reduction in NOTCH1 signalling (> 50% reduction in NOTCH1-ICD, p < 0.05).Gamma secretase inhibitor (GSI), which blocks NOTCH1 signalling, reduced K562/CCN3 colony formation but increased that of K562/control cells. GSI combined with either rCCN3 or imatinib reduced K562 colony formation with enhanced reduction of NOTCH1 signalling observed with combination treatments. We demonstrate an oncogenic role for NOTCH1 in CML and suggest that BCR-ABL disruption of NOTCH1–CCN3 signalling contributes to the pathogenesis of CML.

  9. Systemic overexpression of matricellular protein CCN1 exacerbates obliterative bronchiolitis in mouse tracheal allografts.

    PubMed

    Raissadati, Alireza; Nykänen, Antti I; Tuuminen, Raimo; Syrjälä, Simo O; Krebs, Rainer; Arnaudova, Ralica; Rouvinen, Eeva; Wang, Xiaomin; Poller, Wolfgang; Lemström, Karl B

    2015-12-01

    Obliterative bronchiolitis (OB) involves airway epithelial detachment, fibroproliferation, and inflammation, resulting in chronic rejection and transplant failure. Cysteine-rich 61 (CCN1) is an integrin receptor antagonist with a context-dependent role in inflammatory and fibroproliferative processes. We used a mouse tracheal OB model to investigate the role of CCN1 in the development of lung allograft OB. C57Bl/6 mice received a systemic injection of CCN1-expressing adenoviral vectors 2 days prior to subcutaneous implantation of tracheal allografts from major MHC-mismatched BALB/c mice. We treated another group of tracheal allograft recipients with cyclic arginine-glycine-aspartic acid peptide to dissect the role of αvβ3-integrin signaling in mediating CCN1 effects in tracheal allografts. Allografts were removed 4 weeks after transplantation and analyzed for luminal occlusion, inflammation, and vasculogenesis. CCN1 overexpression induced luminal occlusion (P < 0.05), fibroproliferation, and smooth muscle cell proliferation (P < 0.05). Selective activation of αvβ3-integrin receptor failed to mimic the actions of CCN1, and blocking failed to inhibit the effects of CCN1 in tracheal allografts. In conclusion, CCN1 exacerbates tracheal OB by enhancing fibroproliferation via an αvβ3-integrin-independent pathway. Further experiments are required to uncover its potentially harmful role in the development of OB after lung transplantation.

  10. The matricellular protein CCN1 mediates neutrophil efferocytosis in cutaneous wound healing.

    PubMed

    Jun, Joon-Il; Kim, Ki-Hyun; Lau, Lester F

    2015-06-16

    Neutrophil infiltration constitutes the first step in wound healing, although their timely clearance by macrophage engulfment, or efferocytosis, is critical for efficient tissue repair. However, the specific mechanism for neutrophil clearance in wound healing remains undefined. Here we uncover a key role for CCN1 in neutrophil efferocytosis by acting as a bridging molecule that binds phosphatidylserine, the 'eat-me' signal on apoptotic cells and integrins αvβ3/αvβ5 in macrophages to trigger efferocytosis. Both knockin mice expressing a mutant CCN1 that is unable to bind αvβ3/αvβ5 and mice with Ccn1 knockdown are defective in neutrophil efferocytosis, resulting in exuberant neutrophil accumulation and delayed healing. Treatment of wounds with CCN1 accelerates neutrophil clearance in both Ccn1 knockin mice and diabetic Lepr(db/db) mice, which suffer from neutrophil persistence and impaired healing. These findings establish CCN1 as a critical opsonin in skin injury and suggest a therapeutic potential for CCN1 in certain types of non-healing wounds.

  11. Structural basis for substrate discrimination and integrin binding by autotaxin

    PubMed Central

    Hausmann, Jens; Kamtekar, Satwik; Christodoulou, Evangelos; Day, Jacqueline E.; Wu, Tao; Fulkerson, Zachary; Albers, Harald M.H.G.; van Meeteren, Laurens A.; Houben, Anna; van Zeijl, Leonie; Jansen, Silvia; Andries, Maria; Hall, Troii; Pegg, Lyle E.; Benson, Timothy E.; Kasiem, Mobien; Harlos, Karl; Vander Kooi, Craig; Smyth, Susan S.; Ovaa, Huib; Bollen, Mathieu; Morris, Andrew J.; Moolenaar, Wouter H.; Perrakis, Anastassis

    2010-01-01

    Autotaxin (ATX) or ecto-nucleotide pyrophosphatase/phosphodiesterase-2 (ENPP2) is a secreted lysophospholipase D that generates the lipid mediator lysophosphatidic acid (LPA), a mitogen and chemo-attractant for many cell types. ATX-LPA signaling has roles in various pathologies including tumour progression and inflammation. However, the molecular basis of substrate recognition and catalysis, and the mechanism of interaction with target cells, has been elusive. Here we present the crystal structure of ATX, alone and in complex with a small-molecule inhibitor. We identify a hydrophobic lipid-binding pocket and map key residues required for catalysis and selection between nucleotide and phospholipid substrates. We show that ATX interacts with cell-surface integrins via its N-terminal somatomedin-B-like domains, using an atypical mechanism. Our results define determinants of substrate discrimination by the ENPP family, suggest how ATX promotes localized LPA signaling, and enable new approaches to target ATX with small-molecule therapeutics. PMID:21240271

  12. Structural basis of substrate discrimination and integrin binding by autotaxin

    SciTech Connect

    Hausmann, Jens; Kamtekar, Satwik; Christodoulou, Evangelos; Day, Jacqueline E.; Wu, Tao; Fulkerson, Zachary; Albers, Harald M.H.G.; van Meeteren, Laurens A.; Houben, Anna J.S.; van Zeijl, Leonie; Jansen, Silvia; Andries, Maria; Hall, Troii; Pegg, Lyle E.; Benson, Timothy E.; Kasiem, Mobien; Harlos, Karl; Vander Kooi, Craig W.; Smyth, Susan S.; Ovaa, Huib; Bollen, Mathieu; Morris, Andrew J.; Moolenaar, Wouter H.; Perrakis, Anastassis

    2013-09-25

    Autotaxin (ATX, also known as ectonucleotide pyrophosphatase/phosphodiesterase-2, ENPP2) is a secreted lysophospholipase D that generates the lipid mediator lysophosphatidic acid (LPA), a mitogen and chemoattractant for many cell types. ATX-LPA signaling is involved in various pathologies including tumor progression and inflammation. However, the molecular basis of substrate recognition and catalysis by ATX and the mechanism by which it interacts with target cells are unclear. Here, we present the crystal structure of ATX, alone and in complex with a small-molecule inhibitor. We have identified a hydrophobic lipid-binding pocket and mapped key residues for catalysis and selection between nucleotide and phospholipid substrates. We have shown that ATX interacts with cell-surface integrins through its N-terminal somatomedin B-like domains, using an atypical mechanism. Our results define determinants of substrate discrimination by the ENPP family, suggest how ATX promotes localized LPA signaling and suggest new approaches for targeting ATX with small-molecule therapeutic agents.

  13. Matricellular TSP-1 as a target of interest for impeding melanoma spreading: towards a therapeutic use for TAX2 peptide.

    PubMed

    Jeanne, Albin; Boulagnon-Rombi, Camille; Devy, Jérôme; Théret, Louis; Fichel, Caroline; Bouland, Nicole; Diebold, Marie-Danièle; Martiny, Laurent; Schneider, Christophe; Dedieu, Stéphane

    2016-10-01

    Thrombospondin-1 (TSP-1) is a matricellular glycoprotein known for being highly expressed within a tumor microenvironment, where it promotes an aggressive phenotype particularly by interacting with the CD47 cell-surface receptor. While it originates from the stromal compartment in many malignancies, melanoma is an exception as invasive and metastatic melanoma cells overexpress TSP-1. We recently demonstrated that a new molecular agent that selectively prevents TSP-1 binding to CD47, called TAX2, exhibits anti-cancer properties when administered systemically by decreasing viable tumor tissue within subcutaneous B16 melanoma allografts. At the same time, emerging evidence was published suggesting a contribution of TSP-1 in melanoma metastatic dissemination and resistance to treatment. Through a comprehensive systems biology approach based on multiple genomics and proteomics databases analyses, we first identified a TSP-1-centered interaction network that is overexpressed in metastatic melanoma. Then, we investigated the effects of disrupting TSP-1:CD47 interaction in A375 human malignant melanoma xenografts. In this model, TAX2 systemic administrations induce tumor necrosis by decreasing intra-tumoral blood flow, while concomitantly making tumors less infiltrative. Besides, TAX2 treatment also drastically inhibits B16F10 murine melanoma cells metastatic dissemination and growth in a syngeneic experimental model of lung metastasis, as demonstrated by histopathological analyses as well as longitudinal and quantitative µCT follow-up of metastatic progression. Altogether, the results obtained by combining bioinformatics and preclinical studies strongly suggest that targeting TSP-1/CD47 axis may represent a valuable therapeutic alternative for hampering melanoma spreading.

  14. The Elastin Receptor Complex: A Unique Matricellular Receptor with High Anti-tumoral Potential

    PubMed Central

    Scandolera, Amandine; Odoul, Ludivine; Salesse, Stéphanie; Guillot, Alexandre; Blaise, Sébastien; Kawecki, Charlotte; Maurice, Pascal; El Btaouri, Hassan; Romier-Crouzet, Béatrice; Martiny, Laurent; Debelle, Laurent; Duca, Laurent

    2016-01-01

    Elastin, one of the longest-lived proteins, confers elasticity to tissues with high mechanical constraints. During aging or pathophysiological conditions such as cancer progression, this insoluble polymer of tropoelastin undergoes an important degradation leading to the release of bioactive elastin-derived peptides (EDPs), named elastokines. EDP exhibit several biological functions able to drive tumor development by regulating cell proliferation, invasion, survival, angiogenesis, and matrix metalloproteinase expression in various tumor and stromal cells. Although, several receptors have been suggested to bind elastokines (αvβ3 and αvβ5 integrins, galectin-3), their main receptor remains the elastin receptor complex (ERC). This heterotrimer comprises a peripheral subunit, named elastin binding protein (EBP), associated to the protective protein/cathepsin A (PPCA). The latter is bound to a membrane-associated protein called Neuraminidase-1 (Neu-1). The pro-tumoral effects of elastokines have been linked to their binding onto EBP. Additionally, Neu-1 sialidase activity is essential for their signal transduction. Consistently, EDP-EBP interaction and Neu-1 activity emerge as original anti-tumoral targets. Interestingly, besides its direct involvement in cancer progression, the ERC also regulates diabetes outcome and thrombosis, an important risk factor for cancer development and a vascular process highly increased in patients suffering from cancer. In this review, we will describe ERC and elastokines involvement in cancer development suggesting that this unique receptor would be a promising therapeutic target. We will also discuss the pharmacological concepts aiming at blocking its pro-tumoral activities. Finally, its emerging role in cancer-associated complications and pathologies such as diabetes and thrombotic events will be also considered. PMID:26973522

  15. The Elastin Receptor Complex: A Unique Matricellular Receptor with High Anti-tumoral Potential.

    PubMed

    Scandolera, Amandine; Odoul, Ludivine; Salesse, Stéphanie; Guillot, Alexandre; Blaise, Sébastien; Kawecki, Charlotte; Maurice, Pascal; El Btaouri, Hassan; Romier-Crouzet, Béatrice; Martiny, Laurent; Debelle, Laurent; Duca, Laurent

    2016-01-01

    Elastin, one of the longest-lived proteins, confers elasticity to tissues with high mechanical constraints. During aging or pathophysiological conditions such as cancer progression, this insoluble polymer of tropoelastin undergoes an important degradation leading to the release of bioactive elastin-derived peptides (EDPs), named elastokines. EDP exhibit several biological functions able to drive tumor development by regulating cell proliferation, invasion, survival, angiogenesis, and matrix metalloproteinase expression in various tumor and stromal cells. Although, several receptors have been suggested to bind elastokines (αvβ3 and αvβ5 integrins, galectin-3), their main receptor remains the elastin receptor complex (ERC). This heterotrimer comprises a peripheral subunit, named elastin binding protein (EBP), associated to the protective protein/cathepsin A (PPCA). The latter is bound to a membrane-associated protein called Neuraminidase-1 (Neu-1). The pro-tumoral effects of elastokines have been linked to their binding onto EBP. Additionally, Neu-1 sialidase activity is essential for their signal transduction. Consistently, EDP-EBP interaction and Neu-1 activity emerge as original anti-tumoral targets. Interestingly, besides its direct involvement in cancer progression, the ERC also regulates diabetes outcome and thrombosis, an important risk factor for cancer development and a vascular process highly increased in patients suffering from cancer. In this review, we will describe ERC and elastokines involvement in cancer development suggesting that this unique receptor would be a promising therapeutic target. We will also discuss the pharmacological concepts aiming at blocking its pro-tumoral activities. Finally, its emerging role in cancer-associated complications and pathologies such as diabetes and thrombotic events will be also considered.

  16. BMP9 Crosstalk with the Hippo Pathway Regulates Endothelial Cell Matricellular and Chemokine Responses

    PubMed Central

    Young, Kira; Tweedie, Eric; Conley, Barbara; Ames, Jacquelyn; FitzSimons, MaryLynn; Brooks, Peter; Liaw, Lucy; Vary, Calvin P. H.

    2015-01-01

    Endoglin is a type III TGFβ auxiliary receptor that is upregulated in endothelial cells during angiogenesis and, when mutated in humans, results in the vascular disease hereditary hemorrhagic telangiectasia (HHT). Though endoglin has been implicated in cell adhesion, the underlying molecular mechanisms are still poorly understood. Here we show endoglin expression in endothelial cells regulates subcellular localization of zyxin in focal adhesions in response to BMP9. RNA knockdown of endoglin resulted in mislocalization of zyxin and altered formation of focal adhesions. The mechanotransduction role of focal adhesions and their ability to transmit regulatory signals through binding of the extracellular matrix are altered by endoglin deficiency. BMP/TGFβ transcription factors, SMADs, and zyxin have recently been implicated in a newly emerging signaling cascade, the Hippo pathway. The Hippo transcription coactivator, YAP1 (yes-associated protein 1), has been suggested to play a crucial role in mechanotransduction and cell-cell contact. Identification of BMP9-dependent nuclear localization of YAP1 in response to endoglin expression suggests a mechanism of crosstalk between the two pathways. Suppression of endoglin and YAP1 alters BMP9-dependent expression of YAP1 target genes CCN1 (cysteine-rich 61, CYR61) and CCN2 (connective tissue growth factor, CTGF) as well as the chemokine CCL2 (monocyte chemotactic protein 1, MCP-1). These results suggest a coordinate effect of endoglin deficiency on cell matrix remodeling and local inflammatory responses. Identification of a direct link between the Hippo pathway and endoglin may reveal novel mechanisms in the etiology of HHT. PMID:25909848

  17. BMP9 Crosstalk with the Hippo Pathway Regulates Endothelial Cell Matricellular and Chemokine Responses.

    PubMed

    Young, Kira; Tweedie, Eric; Conley, Barbara; Ames, Jacquelyn; FitzSimons, MaryLynn; Brooks, Peter; Liaw, Lucy; Vary, Calvin P H

    2015-01-01

    Endoglin is a type III TGFβ auxiliary receptor that is upregulated in endothelial cells during angiogenesis and, when mutated in humans, results in the vascular disease hereditary hemorrhagic telangiectasia (HHT). Though endoglin has been implicated in cell adhesion, the underlying molecular mechanisms are still poorly understood. Here we show endoglin expression in endothelial cells regulates subcellular localization of zyxin in focal adhesions in response to BMP9. RNA knockdown of endoglin resulted in mislocalization of zyxin and altered formation of focal adhesions. The mechanotransduction role of focal adhesions and their ability to transmit regulatory signals through binding of the extracellular matrix are altered by endoglin deficiency. BMP/TGFβ transcription factors, SMADs, and zyxin have recently been implicated in a newly emerging signaling cascade, the Hippo pathway. The Hippo transcription coactivator, YAP1 (yes-associated protein 1), has been suggested to play a crucial role in mechanotransduction and cell-cell contact. Identification of BMP9-dependent nuclear localization of YAP1 in response to endoglin expression suggests a mechanism of crosstalk between the two pathways. Suppression of endoglin and YAP1 alters BMP9-dependent expression of YAP1 target genes CCN1 (cysteine-rich 61, CYR61) and CCN2 (connective tissue growth factor, CTGF) as well as the chemokine CCL2 (monocyte chemotactic protein 1, MCP-1). These results suggest a coordinate effect of endoglin deficiency on cell matrix remodeling and local inflammatory responses. Identification of a direct link between the Hippo pathway and endoglin may reveal novel mechanisms in the etiology of HHT.

  18. Dentin Matrix Protein-1 Isoforms Promote Differential Cell Attachment and Migration*S⃞

    PubMed Central

    von Marschall, Zofia; Fisher, Larry W.

    2008-01-01

    Dentin matrix protein-1 (DMP1), bone sialoprotein (BSP), and osteopontin (OPN) are three SIBLINGs (small integrin-binding ligand, N-linked glycoproteins) co-expressed/secreted by skeletal and active ductal epithelial cells. Although etiological mechanisms remain unclear, DMP1 is the only one of these three genes currently known to have mutations resulting in human disease, and yet it remains the least studied. All three contain the highly conserved integrin-binding tripeptide, RGD, and experiments comparing the cell attachment and haptotactic migration-enhancing properties of DMP1 to BSP and OPN were performed using human skeletal (MG63 and primary dental pulp cells) and salivary gland (HSG) cells. Mutation of any SIBLING's RGD destroyed all attachment and migration activity. Using itsαVβ5 integrin, HSG cells attached to BSP but not to DMP1 or OPN. However, HSG cells could not migrate onto BSP in a modified Boyden chamber assay. Expression of αVβ3 integrin enhanced HSG attachment to DMP1 and OPN and promoted haptotactic migration onto all three proteins. Interchanging the first four coding exons or the conserved amino acids adjacent to the RGD of DMP1 with corresponding sequences of BSP did not enhance the ability of DMP1 to bindαVβ5. For αVβ3-expressing cells, intact DMP1, its BMP1-cleaved C-terminal fragment, and exon six lacking all post-translational modifications worked equally well but the proteoglycan isoform of DMP1 had greatly reduced ability for cell attachment and migration. The sequence specificity of the proposed BMP1-cleavage site of DMP1 was verified by mutation analysis. Direct comparison of the three proteins showed that cells discriminate among these SIBLINGs and among DMP1 isoforms. PMID:18819913

  19. Cell surface receptors for CCN proteins.

    PubMed

    Lau, Lester F

    2016-06-01

    The CCN family (CYR61; CTGF; NOV; CCN1-6; WISP1-3) of matricellular proteins in mammals is comprised of six homologous members that play important roles in development, inflammation, tissue repair, and a broad range of pathological processes including fibrosis and cancer. Despite considerable effort to search for a high affinity CCN-specific receptor akin to growth factor receptors, no such receptor has been found. Rather, CCNs bind several groups of multi-ligand receptors as characteristic of other matricellular proteins. The most extensively documented among CCN-binding receptors are integrins, including αvβ3, αvβ5, α5β1, α6β1, αIIbβ3, αMβ2, and αDβ2, which mediate diverse CCN functions in various cell types. CCNs also bind cell surface heparan sulfate proteoglycans (HSPGs), low density liproprotein receptor-related proteins (LRPs), and the cation-independent mannose-6-phosphate (M6P) receptor, which are endocytic receptors that may also serve as co-receptors in cooperation with other cell surface receptors. CCNs have also been reported to bind FGFR-2, Notch, RANK, and TrkA, potentially altering the affinities of these receptors for their ligands. The ability of CCNs to bind a multitude of receptors in various cell types may account for the remarkable versatility of their functions, and underscore the diverse signaling pathways that mediate their activities.

  20. Taking Aim at the Extracellular Matrix: CCN Proteins as Emerging Therapeutic Targets

    PubMed Central

    Jun, Joon-Il; Lau, Lester F.

    2013-01-01

    The CCN family of matricellular proteins is critical for embryonic development and plays important roles in inflammation, wound healing, and injury repair in the adult. Deregulation of their expression or activities contributes to the pathobiology of myriad diseases, many of which may arise when inflammation or tissue injury becomes chronic, including fibrosis, arthrosclerosis, arthritis, diabetic nephropathy and retinopathy, and cancer. Emerging studies indicate that targeting CCN expression or signaling pathways holds promise in the development of diagnostics and therapeutics for such diseases. This review summarizes the biology of CCN proteins, their roles in various pathologies, and potential as therapeutic targets. PMID:22129992

  1. Chronic Wound Dressings Based on Collagen-Mimetic Proteins

    PubMed Central

    Cereceres, Stacy; Touchet, Tyler; Browning, Mary Beth; Smith, Clayton; Rivera, Jose; Höök, Magnus; Whitfield-Cargile, Canaan; Russell, Brooke; Cosgriff-Hernandez, Elizabeth

    2015-01-01

    Objective: Chronic wounds are projected to reach epidemic proportions due to the aging population and the increasing incidence of diabetes. There is a strong clinical need for an improved wound dressing that can balance wound moisture, promote cell migration and proliferation, and degrade at an appropriate rate to minimize the need for dressing changes. Approach: To this end, we have developed a bioactive, hydrogel microsphere wound dressing that incorporates a collagen-mimetic protein, Scl2GFPGER, to promote active wound healing. A redesigned Scl2GFPGER, engineered collagen (eColGFPGER), was created to reduce steric hindrance of integrin-binding motifs and increase overall stability of the triple helical backbone, thereby resulting in increased cell adhesion to substrates. Results: This study demonstrates the successful modification of the Scl2GFPGER protein to eColGFPGER, which displayed enhanced stability and integrin interactions. Fabrication of hydrogel microspheres provided a matrix with adaptive moisture technology, and degradation rates have potential for use in human wounds. Innovation: This collagen-mimetic wound dressing was designed to permit controlled modulation of cellular interactions and degradation rate without impact on other physical properties. Its fabrication into uniform hydrogel microspheres provides a bioactive dressing that can readily conform to irregular wounds. Conclusion: Overall, this new eColGFPGER shows strong promise in the generation of bioactive hydrogels for wound healing as well as a variety of tissue scaffolds. PMID:26244101

  2. Molecular cloning and characterisation of a pattern recognition protein, lipopolysaccharide and beta-1,3-glucan binding protein (LGBP) from Chinese shrimp Fenneropenaeus chinensis.

    PubMed

    Liu, Fengsong; Li, Fuhua; Dong, Bo; Wang, Xiaomei; Xiang, Jianhai

    2009-03-01

    A pattern recognition protein (PRP), lipopolysaccharide and beta-1,3-glucan binding protein (LGBP) cDNA was cloned from the haemocyte of Chinese shrimp Fenneropenaeus chinensis by the techniques of homology cloning and RACE. Analysis of nucleotide sequence revealed that the full-length cDNA of 1,275 bp has an open reading frame of 1,098 bp encoding a protein of 366 amino acids including a 17 amino acid signal peptide. Sequence comparison of the deduced amino acid sequence of F. chinensis LGBP showed a high identity of 94%, 90%, 87%, 72% and 63% with Penaeus monodon BGBP, Litopenaeus stylirostris LGBP, Marsupenaeu japonicus BGBP, Homarus gammarus BGBP and Pacifastacus leniusculus LGBP, respectively. The calculated molecular mass of the mature protein is 39,857 Da with a deduced pI of 4.39. Two putative integrin binding motifs, RGD (Arg-Gly-Asp) and a potential recognition motif for beta-1,3-linkage of polysaccharides were observed in LGBP sequence. RT-PCR analysis showed that LGBP gene expresses in haemocyte and hepatopancreas only, but not in other tissues. Capillary electrophoresis RT-PCR method was used to quantify the variation of mRNA transcription level during artificial infection with heat-killed Vibrio anguillarum and Staphylococcus aureusin. A significant enhancement of LGBP transcription was appeared at 6 h post-injection in response to bacterial infection. These results have provided useful information to understand the function of LGBP in shrimp. PMID:18163220

  3. Identification, characterization, and expression of dentin matrix protein 1 gene in Xenopus laevis.

    PubMed

    Yonekura, Tomoko; Homma, Hiromi; Sakurai, Atsuo; Moriguchi, Mitsuko; Miake, Yasuo; Toyosawa, Satoru; Shintani, Seikou

    2013-12-01

    Dentin matrix protein 1 (DMP1) is an acidic extracellular matrix protein expressed mainly in bone and dentin, and is a member of the small integrin-binding ligand N-linked glycoprotein (SIBLING) family. The DMP1 gene, however, appears to evolve rapidly in comparison with other SIBLING genes, even though such functionally important molecules usually evolve more slowly than less important ones. The purpose of this study was to identify and characterize an ortholog of the DMP1 gene in an amphibian (Xenopus laevis; X. laevis) to clarify molecular evolutionary alterations in DMP1 associated with calcified tissues in tetrapods. Furthermore, we analyzed the mRNA expression of this gene to elucidate its functional change in bone and developing tooth germ in comparison with amniote DMP1s. The similarities of the deduced amino acid sequence of X. laevis DMP1 to that of the corresponding amniote proteins were low, although they did share several unique features specific to DMP1 and have similar properties. Expression of X. laevis DMP1 mRNA was predominant in osteocytes and odontoblasts, but only transiently observed in ameloblasts, as in amniotes. These results suggest that DMP1 has conserved several functions during tetrapod evolution. This indicates that continuity of biochemical properties has been more important in maintaining DMP1 functionality than that of the sequence of amino acid residues, which has undergone change over the course of molecular evolution.

  4. The RGD integrin binding site in human L1-CAM is important for nuclear signaling

    SciTech Connect

    Gast, Daniela; Riedle, Svenja; Kiefel, Helena; Mueerkoester, Susanne Sebens; Schaefer, Heiner; Schaefer, Michael K.E.; Altevogt, Peter

    2008-08-01

    L1 cell adhesion molecule (L1-CAM) is a transmembrane cell adhesion molecule initially defined as a promigratory molecule in the developing nervous system. L1 is also overexpressed in a variety of human carcinomas and is associated with bad prognosis. In carcinoma cell lines L1 augments cell motility and metastasis, tumor growth in nude mice and induces expression of L1-dependent genes. It is not known whether L1-signaling requires ligand binding. The RGD motif in the sixth Ig domain of L1 is a binding site for integrins. In the present study we analyzed the role of RGDs in L1-signaling using site-directed mutagenesis combined with antibody blocking studies. We observed that L1-RGE expressing HEK293 cells showed reduced cell-cell binding, cell motility, invasiveness and tumor growth in NOD/SCID mice. The RGE-mutation impaired L1-dependent gene regulation and antibodies to {alpha}v{beta}5 integrin had similar effects. Mutant L1 was unable to translocate to the nucleus. Our findings highlight the importance of the RGD site in L1 for human tumors and suggest that nuclear signaling of L1 is dependent on integrins.

  5. Proteins.

    ERIC Educational Resources Information Center

    Doolittle, Russell F.

    1985-01-01

    Examines proteins which give rise to structure and, by virtue of selective binding to other molecules, make genes. Binding sites, amino acids, protein evolution, and molecular paleontology are discussed. Work with encoding segments of deoxyribonucleic acid (exons) and noncoding stretches (introns) provides new information for hypotheses. (DH)

  6. Protein

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Proteins are the major structural and functional components of all cells in the body. They are macromolecules that comprise 1 or more chains of amino acids that vary in their sequence and length and are folded into specific 3-dimensional structures. The sizes and conformations of proteins, therefor...

  7. Protease-Triggered, Integrin-Targeted Cellular Uptake of Recombinant Protein Micelles.

    PubMed

    Gao, Chen; Vargo, Kevin B; Hammer, Daniel A

    2016-09-01

    Targeting nanoparticles for drug delivery has great potential for improving efficacy and reducing side effects from systemic toxicity. New developments in the assembly of materials afford the opportunity to expose cryptic targeting domains in tissue-specific microenvironments in which certain proteases are expressed. Here, recombinant proteins are designed to combine the responsiveness to environmental proteases with specific targeting. Materials made recombinantly allow complete control over amino acid sequence, in which each molecule is identically functionalized. Previously, oleosin, a naturally occurring plant protein that acts as a surfactant, has been engineered to self-assemble into spherical micelles-a useful structure for drug delivery. To make oleosins that are locally activated to bind receptors, oleosin is genetically modified to incorporate the integrin-binding motif RGDS just behind a domain cleavable by thrombin. The resulting modified oleosin self-assembles into spherical micelles in aqueous environments, with the RGDS motif protected by the thrombin-cleavable domain. Upon the addition of thrombin, the RGDS is exposed and the binding of the spherical micelles to breast cancer cells is increased fourfold. PMID:27284959

  8. SED1/MFG-E8: a bi-motif protein that orchestrates diverse cellular interactions.

    PubMed

    Raymond, Adam; Ensslin, Michael A; Shur, Barry D

    2009-04-15

    MFG-E8 was initially identified as a principle component of the Milk Fat Globule, a membrane-encased collection of proteins and triglycerides that bud from the apical surface of mammary epithelia during lactation. It has since been independently identified in many species and by many investigators and given a variety of names, including p47, lactadherin, rAGS, PAS6/7, and BA-46. The acronym SED1 was proposed to bring cohesion to this nomenclature based upon it being a Secreted protein that contains two distinct functional domains: an N-terminal domain with two EGF-repeats, the second of which has an integrin-binding RGD motif, and a C-terminal domain with two Discoidin/F5/8C domains that bind to anionic phospholipids and/or extracellular matrices. SED1/MFG-E8 is now known to participate in a wide variety of cellular interactions, including phagocytosis of apoptotic lymphocytes and other apoptotic cells, adhesion between sperm and the egg coat, repair of intestinal mucosa, mammary gland branching morphogenesis, angiogenesis, among others. This article will explore the various roles proposed for SED1/MFG-E8, as well as its provocative therapeutic potential. PMID:19204935

  9. Structural and functional determinants in adenovirus type 2 penton base recombinant protein.

    PubMed Central

    Karayan, L; Hong, S S; Gay, B; Tournier, J; d'Angeac, A D; Boulanger, P

    1997-01-01

    Discrete domains involved in structural and functional properties of adenovirus type 2 (Ad2) penton base were investigated with site-directed mutagenesis of the recombinant protein expressed in baculovirus-infected cells. Seventeen substitution mutants were generated and phenotyped for various functions in insect and human cells as follows. (i) Pentamerization of the penton base protein was found to be dependent on three amino acid side chains, the indole ring of Trp119, the hydroxylic group of Tyr553, and the basic group of Lys556. (ii) Arg254, Cys432, and Trp439, the stretch of basic residues at positions 547 to 556, and Arg340 of the RGD motif played a critical role in stable fiber-penton base interactions in vivo. (iii) Nuclear localization of penton base in Sf9 cells was negatively affected in mutants W119H or W165H, and, to a lesser extent, by substitutions in the consensus polybasic signal at positions 547 to 549. (iv) Penton base mutants were also assayed for HeLa cell binding, cell detachment, plasmid DNA internalization, and Ad-mediated gene delivery. The results obtained suggested that the previously identified integrin-binding motifs RGD340 and LDV287 were functionally and/or topologically related to other discrete regions which include Trp119, Trp165, Cys246, Cys432, and Trp439, all of which were involved in penton base-cell surface recognition, endocytosis, and postendocytotic steps of the virus life cycle. PMID:9343226

  10. Proteins

    NASA Astrophysics Data System (ADS)

    Regnier, Fred E.; Gooding, Karen M.

    Because of the complexity of cellular material and body fluids, it is seldom possible to analyze a natural product directly. Qualitative and quantitative analyses must often be preceded by some purification step that separates the molecular species being examined from interfering materials. In the case of proteins, column liquid chromatography has been used extensively for these fractionations. With the advent of gel permeation, cation exchange, anion exchange, hydrophobic, and affinity chromatography, it became possible to resolve proteins through their fundamental properties of size, charge, hydrophobicity, and biological affinity. The chromatographic separations used in the early isolation and characterization of many proteins later became analytical tools in their routine analysis. Unfortunately, these inherently simple and versatile column chromatographic techniques introduced in the 50s and 60s have a severe limitation in routine analysis-separation time. It is common to encounter 1-24 h separation times with the classical gel-type supports.

  11. p73 promotes glioblastoma cell invasion by directly activating POSTN (periostin) expression

    PubMed Central

    Landré, Vivien; Antonov, Alexey; Knight, Richard; Melino, Gerry

    2016-01-01

    Glioblastoma Multiforme is one of the most highly metastatic cancers and constitutes 70% of all gliomas. Despite aggressive treatments these tumours have an exceptionally bad prognosis, mainly due to therapy resistance and tumour recurrence. Here we show that the transcription factor p73 confers an invasive phenotype by directly activating expression of POSTN (periostin, HGNC:16953) in glioblastoma cells. Knock down of endogenous p73 reduces invasiveness and chemo-resistance, and promotes differentiation in vitro. Using chromatin immunoprecipitation and reporter assays we demonstrate that POSTN, an integrin binding protein that has recently been shown to play a major role in metastasis, is a transcriptional target of TAp73. We further show that POSTN overexpression is sufficient to rescue the invasive phenotype of glioblastoma cells after p73 knock down. Additionally, bioinformatics analysis revealed that an intact p73/POSTN axis, where POSTN and p73 expression is correlated, predicts bad prognosis in several cancer types. Taken together, our results support a novel role of TAp73 in controlling glioblastoma cell invasion by regulating the expression of the matricellular protein POSTN. PMID:26930720

  12. Female-Specific Induction of Rat Pituitary Dentin Matrix Protein-1 by GnRH

    PubMed Central

    Kucka, Marek; Bjelobaba, Ivana; Clokie, Samuel J. H.; Klein, David C.

    2013-01-01

    Hypothalamic GnRH is the primary regulator of reproduction in vertebrates, acting via the G protein-coupled GnRH receptor (GnRHR) in pituitary gonadotrophs to control synthesis and release of gonadotropins. To identify elements of the GnRHR-coupled gene network, GnRH was applied in a pulsatile manner for 6 hours to a mixed population of perifused pituitary cells from cycling females, mRNA was extracted, and RNA sequencing analysis was performed. This revealed 83 candidate-regulated genes, including a large number coding for secreted proteins. Most notably, GnRH induces a greater than 600-fold increase in expression of dentin matrix protein-1 (Dmp1), one of five members of the small integrin-binding ligand N-linked glycoprotein gene family. The Dmp1 response is mediated by the GnRHR, not elicited by other hypothalamic releasing factors, and is approximately 20-fold smaller in adult male pituitary cells. The sex-dependent Dmp1 response is established during the peripubertal period and independent of the developmental pattern of Gnrhr expression. In vitro, GnRH-induced expression of this gene is coupled with release of DMP1 in extracellular medium through the regulated secretory pathway. In vivo, pituitary Dmp1 expression in identified gonadotrophs is elevated after ovulation. Cell signaling studies revealed that the GnRH induction of Dmp1 is mediated by the protein kinase C signaling pathway and reflects opposing roles of ERK1/2 and p38 MAPK; in addition, the response is facilitated by progesterone. These results establish that DMP1 is a novel secretory protein of female rat gonadotrophs, the synthesis and release of which are controlled by the hypothalamus through the GnRHR signaling pathway. This advance raises intriguing questions about the intrapituitary and downstream effects of this new player in GnRH signaling. PMID:24085820

  13. Weaponizing human EGF-containing fibulin-like extracellular matrix protein 1 (EFEMP1) for 21st century cancer therapeutics

    PubMed Central

    Zhou, Yi-Hong; Hu, Yuanjie; Yu, Liping; Ke, Chao; Vo, Christopher; Hsu, Hao; Li, Zhenzhi; Di Donato, Anne T.; Chaturbedi, Abhishek; Hwang, Ji Won; Siegel, Eric R.; Linskey, Mark E.

    2016-01-01

    De-regulated EFEMP1 gene expression in solid tumors has been widely reported with conflicting roles. We dissected EFEMP1 to identify domains responsible for its cell context-dependent dual functions, with the goal being to construct an EFEMP1-derived tumor-suppressor protein (ETSP) that lacked tumor-promoting function. Exon/intron boundaries of EFEMP1 were used as boundaries of functional modules in constructing EFEMP1 variants, with removal of various module(s), and/or mutating an amino acid residue to convert a weak integrin binding-site into a strong one. A series of in vitro assays on cancerous features, and subcutaneous and intracranial xenograft-formation assays, were carried out for effects from overexpression of wild-type and variant forms of EFEMP1 in two glioma subpopulations characterized as tumor mass-forming cells (TMCs) or stem-like tumor initiating cells (STICs), where EFEMP1 showed cellcontext- dependent dual functions. One of the EFEMP1 variants was identified as the sought-after ETSP, which had a stronger tumor-suppression function in TMCs by targeting EGFR and angiogenesis, and a new tumor-suppression function in STICs by targeting NOTCH signaling and MMP2-mediated cell invasion. Therefore, ETSP may form the basis for further important research to develop a novel cancer therapy to treat many types of cancer by its tumor suppressor effect in the extracellular matrix compartment. PMID:27713911

  14. Biologically engineered protein-graft-poly(ethylene glycol) hydrogels: A cell-adhesive and plasmin-degradable biosynthetic material for tissue repair

    NASA Astrophysics Data System (ADS)

    Halstenberg, Sven

    2002-01-01

    The goal of the research presented in this dissertation was to create a biomimetic artificial material that exhibits functions of extracellular matrix relevant for improved nerve regeneration. Neural adhesion peptides were photoimmobilized on highly crosslinked poly(ethylene glycol)-based substrates that were otherwise non-adhesive. Neurons adhered in two-dimensional patterns for eleven hours, but no neurites extended. To enable neurite extension and nerve regeneration in three dimensions, and to address the need for specifically cell adhesive and cell degradable materials for clinical applications in tissue repair in general, an artificial protein was recombinantly expressed and purified that consisted of a repeating amino acid sequence based on fibrinogen and anti-thrombin III. The recombinant protein contained integrin-binding RGD sites, plasmin degradation sites, heparin binding sites, and six thiol-containing cysteine residues as grafting sites for poly(ethylene glycol) diacrylate via Michael-type conjugate addition. The resulting protein-graft-poly(ethylene glycol)acrylates were crosslinked by photopolymerization to form hydrogels. Although three-dimensional, RGD mediated and serine protease-dependent ingrowth of human fibroblasts into protein-graft-poly(ethylene glycol) hydrogels occurred, only surface neurite outgrowth was observed from chick dorsal root ganglia. Axonal outgrowth depended on the concentration of matrix-bound heparin, suggesting that improved mechanical strength of the hydrogels and possible immobilization of neuroactive factors due to the presence of heparin promoted neurite outgrowth. Together, the above results show that specific biological functions can be harnessed by protein-graft-poly(ethylene glycol) hydrogels to serve as matrices for tissue repair and regeneration. In particular, the two design objectives, specific cell adhesion and degradability by cell-associated proteases, were fulfilled by the material. In the future, this and

  15. Crystal Structure of the N-terminal Domain of the Group B Streptococcus Alpha C Protein

    SciTech Connect

    Auperin,T.; Bolduc, G.; Baron, M.; Heroux, A.; Filman, D.; Madoff, L.; Hogle, J.

    2005-01-01

    Group B Streptococcus (GBS) is the leading cause of bacterial pneumonia, sepsis, and meningitis among neonates and an important cause of morbidity among pregnant women and immunocompromised adults. Invasive diseases due to GBS are attributed to the ability of the pathogen to translocate across human epithelial surfaces. The alpha C protein (ACP) has been identified as an invasin that plays a role in internalization and translocation of GBS across epithelial cells. The soluble N-terminal domain of ACP (NtACP) blocks the internalization of GBS. We determined the 1.86-{angstrom} resolution crystal structure of NtACP comprising residues Ser{sup 52} through Leu{sup 225} of the full-length ACP. NtACP has two domains, an N-terminal {beta}-sandwich and a C-terminal three-helix bundle. Structural and topological alignments reveal that the {beta}-sandwich shares structural elements with the type III fibronectin fold (FnIII), but includes structural elaborations that make it unique. We have identified a potential integrin-binding motif consisting of Lys-Thr-Asp{sup 146}, Arg{sup 110}, and Asp{sup 118}. A similar arrangement of charged residues has been described in other invasins. ACP shows a heparin binding activity that requires NtACP. We propose a possible heparin-binding site, including one surface of the three-helix bundle, and nearby portions of the sandwich and repeat domains. We have validated this prediction using assays of the heparin binding and cell-adhesion properties of engineered fragments of ACP. This is the first crystal structure of a member of the highly conserved Gram-positive surface alpha-like protein family, and it will enable the internalization mechanism of GBS to be dissected at the atomic level.

  16. SPECT/CT Imaging of High-Risk Atherosclerotic Plaques using Integrin-Binding RGD Dimer Peptides.

    PubMed

    Yoo, Jung Sun; Lee, Jonghwan; Jung, Jae Ho; Moon, Byung Seok; Kim, Soonhag; Lee, Byung Chul; Kim, Sang Eun

    2015-06-30

    Vulnerable atherosclerotic plaques with unique biological signatures are responsible for most major cardiovascular events including acute myocardial infarction and stroke. However, current clinical diagnostic approaches for atherosclerosis focus on anatomical measurements such as the degree of luminal stenosis and wall thickness. An abundance of neovessels with elevated expression of integrin αvβ3 is closely associated with an increased risk of plaque rupture. Herein we evaluated the potential of an αvβ3 integrin-targeting radiotracer, (99m)Tc-IDA-D-[c(RGDfK)]2, for SPECT/CT imaging of high-risk plaque in murine atherosclerosis models. In vivo uptake of (99m)Tc-IDA-D-[c(RGDfK)]2 was significantly higher in atherosclerotic aortas than in relatively normal aortas. Comparison with the negative-control peptide, (99m)Tc-IDA-D-[c(RADfK)]2, proved specific binding of (99m)Tc-IDA-D-[c(RGDfK)]2 for plaque lesions in in vivo SPECT/CT and ex vivo autoradiographic imaging. Histopathological characterization revealed that a prominent SPECT signal of (99m)Tc-IDA-D-[c(RGDfK)]2 corresponded to the presence of high-risk plaques with a large necrotic core, a thin fibrous cap, and vibrant neoangiogenic events. Notably, the RGD dimer based (99m)Tc-IDA-D-[c(RGDfK)]2 showed better imaging performance in comparison with the common monomeric RGD peptide probe (123)I-c(RGDyV) and fluorescence tissue assay corroborated this. Our preclinical data demonstrated that (99m)Tc-IDA-D-[c(RGDfK)]2 SPECT/CT is a sensitive tool to noninvasively gauge atherosclerosis beyond vascular anatomy by assessing culprit plaque neovascularization.

  17. Modeling the Interaction between Integrin-Binding Peptide (RGD) and Rutile Surface: The Effect of Na+ on Peptide Adsorption

    SciTech Connect

    Wu, Chunya; Skelton, Adam; Chen, Mingjun; Vlcek, Lukas; Cummings, Peter T

    2011-01-01

    The dynamics of a single tripeptide Arg-Gly-Asp (RGD) adsorbing onto negatively charged hydroxylated rutile (110) surface in aqueous solution was studied using molecular dynamics (MD) simulations. The results indicate that the adsorbed Na{sup +} ions play an important role in determining the binding geometry of RGD. With an initial 'horseshoe' configuration, the charged side groups (COO{sup -} and NH{sub 2}) of the peptide are able to interact with the surface through direct hydrogen bonds (H bonds) in the very early stage of adsorption. The Na{sup +} ions approach the positively charged Arg side chain, competing with the Arg side chain for adsorption to the negatively charged hydroxyl oxygen. In coordination with the structural adjustment of the peptide, the Arg residue is driven to detach from the rutile surface. In contrast, the Na+ ions in close proximity to the negatively charged Asp side chain contribute to the binding of the COO{sup -} group on the surface, helping the carboxyl oxygen not involved in COO{sup -}-surface H bonds to orientate toward the hydroxyl hydrogens. Once both carboxyl oxygens form enough H bonds with the hydroxyl hydrogens, the redundant ions move toward a more favorable adsorption site.

  18. Modeling the Interaction between Integrin-Binding Peptide (RGD) and Rutile Surface: The Effect of Cation Mediation on Asp Adsorption

    SciTech Connect

    Wu, Chunya; Skelton, Adam; Chen, Mingjun; Vlcek, Lukas; Cummings, Peter T

    2012-01-01

    The binding of a negatively charged residue, aspartic acid (Asp) in tripeptide arginine-glycine-aspartic acid, onto a negatively charged hydroxylated rutile (110) surface in aqueous solution, containing divalent (Mg{sup 2+}, Ca{sup 2+}, or Sr{sup 2+}) or monovalent (Na{sup +}, K{sup +}, or Rb{sup +}) cations, was studied by molecular dynamics (MD) simulations. The results indicate that ionic radii and charges will significantly affect the hydration, adsorption geometry, and distance of cations from the rutile surface, thereby regulating the Asp/rutile binding mode. The adsorption strength of monovalent cations on the rutile surface in the order Na{sup +} > K{sup +} > Rb{sup +} shows a 'reverse' lyotropic trend, while the divalent cations on the same surface exhibit a 'regular' lyotropic behavior with decreasing crystallographic radii (the adsorption strength of divalent cations: Sr{sup 2+} > Ca{sup 2+} > Mg{sup 2+}). The Asp side chain in NaCl, KCl, and RbCl solutions remains stably H-bonded to the surface hydroxyls and the inner-sphere adsorbed compensating monovalent cations act as a bridge between the COO{sup -} group and the rutile, helping to 'trap' the negatively charged Asp side chain on the negatively charged surface. In contrast, the mediating divalent cations actively participate in linking the COO{sup -} group to the rutile surface; thus the Asp side chain can remain stably on the rutile (110) surface, even if it is not involved in any hydrogen bonds with the surface hydroxyls. Inner- and outer-sphere geometries are all possible mediation modes for divalent cations in bridging the peptide to the rutile surface.

  19. Synchronized cell attachment triggered by photo-activatable adhesive ligands allows QCM-based detection of early integrin binding

    PubMed Central

    Iturri, Jagoba; García-Fernández, Luis; Reuning, Ute; García, Andrés J.; Campo, Aránzazu del; Salierno, Marcelo J.

    2015-01-01

    The Quartz Crystal Microbalance with dissipation (QCM-D) technique was applied to monitor and quantify integrin-RGD recognition during the early stages of cell adhesion. Using QCM-D crystals modified with a photo-activatable RGD peptide, the time point of presentation of adhesive ligand at the surface of the QCM-D crystal could be accurately controlled. This allowed temporal resolution of early integrin-RGD binding and the subsequent cell spreading process, and their separate detection by QCM-D. The specificity of the integrin-RGD binding event was corroborated by performing the experiments in the presence of soluble cyclicRGD as a competitor, and cytochalasin D as inhibitor of cell spreading. Larger frequency change in the QCM-D signal was observed for cells with larger spread area, and for cells overexpressing integrin αvβ3 upon stable transfection. This strategy enables quantification of integrin activity which, in turn, may allow discrimination among different cell types displaying distinct integrin subtypes and expression levels thereof. On the basis of these findings, we believe the strategy can be extended to other photoactivatable ligands to characterize cell membrane receptors activity, a relevant issue for cancer diagnosis (and prognosis) as other several pathologies. PMID:25825012

  20. Osteogenic differentiation of human mesenchymal stem cells on α5 integrin binding peptide hydrogels is dependent on substrate elasticity

    PubMed Central

    Gandavarapu, Navakanth R.; Alge, Daniel L.; Anseth, Kristi S.

    2014-01-01

    The extracellular matrix plays a crucial role in controlling human mesenchymal stem cell (hMSC) biology including differentiation, and α5β1 integrin signaling plays an important role during osteogenic differentiation of hMSCs. Here, peptide-functionalized hydrogels were used to examine the role of α5β1 integrin signaling in inducing osteogenic differentiation in hMSCs. Further, the role of substrate elasticity was also studied. A thiolene chemistry was used to functionalize poly(ethylene glycol) hydrogels with a pendant peptide moieity, c(RRETAWA), as previous studies have shown that RRETAWA containing peptides bind to the α5β1 integrin with very high specificity. Notably, hMSC attachment to c(RRETAWA)-functionalized hydrogels was found to occur primarily through α5 integrins, as the number of attached cells was significantly reduced to ~20% upon blocking the α5 integrin during culture. To investigate the interplay between stiffness and c(RRETAWA) concentration, hydrogels were formulated with Young’s moduli of ~2 kPa (soft) and ~25 kPa (stiff) and c(RRETAWA) concentrations of 0.1 mM and 1 mM. Stiff substrates led to ~3.5 fold higher hMSC attachment and ~3 fold higher cell area in comparison to soft substrates. hMSCs formed robust and larger focal adhesions on stiff substrates at 1 mM c(RRETAWA) compared to soft substrates. Alkaline phosphatase (ALP) activity in hMSCs cultured on stiff gels at 0.1 mM and 1 mM c(RRETAWA) was increased 2.5 and 3.5 fold, respectively after 14 days in growth media. hMSCs did not show an increase in ALP activity when cultured on soft gels. Further, gene expression of osteogenic related genes, core binding factor-1, osteopontin and Collagen-1a at day 14 in hMSCs cultured on stiff gels at 1 mM c(RRETAWA) were increased 10, 7 and 4 fold, respectively, while on soft gels, gene expression was at basal levels. Collectively, these results demonstrate that the combination of high substrate stiffness and α5β1 integrin signaling stimulated by c(RRETAWA) is sufficient to induce osteogenic differentiation of hMSCs without requiring the addition of soluble factors. PMID:24660057

  1. SPECT/CT Imaging of High-Risk Atherosclerotic Plaques using Integrin-Binding RGD Dimer Peptides

    PubMed Central

    Sun Yoo, Jung; Lee, Jonghwan; Ho Jung, Jae; Seok Moon, Byung; Kim, Soonhag; Chul Lee, Byung; Eun Kim, Sang

    2015-01-01

    Vulnerable atherosclerotic plaques with unique biological signatures are responsible for most major cardiovascular events including acute myocardial infarction and stroke. However, current clinical diagnostic approaches for atherosclerosis focus on anatomical measurements such as the degree of luminal stenosis and wall thickness. An abundance of neovessels with elevated expression of integrin αvβ3 is closely associated with an increased risk of plaque rupture. Herein we evaluated the potential of an αvβ3 integrin-targeting radiotracer, 99mTc-IDA-D-[c(RGDfK)]2, for SPECT/CT imaging of high-risk plaque in murine atherosclerosis models. In vivo uptake of 99mTc-IDA-D-[c(RGDfK)]2 was significantly higher in atherosclerotic aortas than in relatively normal aortas. Comparison with the negative-control peptide, 99mTc-IDA-D-[c(RADfK)]2, proved specific binding of 99mTc-IDA-D-[c(RGDfK)]2 for plaque lesions in in vivo SPECT/CT and ex vivo autoradiographic imaging. Histopathological characterization revealed that a prominent SPECT signal of 99mTc-IDA-D-[c(RGDfK)]2 corresponded to the presence of high-risk plaques with a large necrotic core, a thin fibrous cap, and vibrant neoangiogenic events. Notably, the RGD dimer based 99mTc-IDA-D-[c(RGDfK)]2 showed better imaging performance in comparison with the common monomeric RGD peptide probe 123I-c(RGDyV) and fluorescence tissue assay corroborated this. Our preclinical data demonstrated that 99mTc-IDA-D-[c(RGDfK)]2 SPECT/CT is a sensitive tool to noninvasively gauge atherosclerosis beyond vascular anatomy by assessing culprit plaque neovascularization. PMID:26123253

  2. Phylogenetic Analysis and Structural Predictions of Human Adenovirus Penton Proteins as a Basis for Tissue-Specific Adenovirus Vector Design▿

    PubMed Central

    Madisch, Ijad; Hofmayer, Soeren; Moritz, Christian; Grintzalis, Alexander; Hainmueller, Jens; Pring-Akerblom, Patricia; Heim, Albert

    2007-01-01

    The penton base is a major capsid protein of human adenoviruses (HAdV) which forms the vertices of the capsid and interacts with hexon and fiber protein. Two hypervariable loops of the penton are exposed on the capsid surface. Sequences of these and 300 adjacent amino acid residues of all 51 HAdV and closely related simian adenoviruses were studied. Adjacent sequences and predicted overall secondary structure were conserved. Phylogenetic analysis revealed clustering corresponding to the HAdV species and recombination events in the origin of HAdV prototypes. All HAdV except serotypes 40 and 41 of species F exhibited an integrin binding RGD motif in the second loop. The lengths of the loops (HVR1 and RGD loops) varied significantly between HAdV species with the longest RGD loop observed in species C and the longest HVR1 in species B. Long loops may permit the insertion of motifs that modify tissue tropism. Genetic analysis of HAdV prime strain p17′H30, a neutralization variant of HAdV-D17, indicated the significance of nonhexon neutralization epitopes for HAdV immune escape. Fourteen highly conserved motifs of the penton base were analyzed by site-directed mutagenesis of HAdV-D8 and tested for sustained induction of early cytopathic effects. Thus, three new motifs essential for penton base function were identified additionally to the RGD site, which interacts with a secondary cellular receptor responsible for internalization. Therefore, our penton primary structure data and secondary structure modeling in combination with the recently published fiber knob sequences may permit the rational design of tissue-specific adenoviral vectors. PMID:17522221

  3. Identification and phylogenetic analysis on lipopolysaccharide and beta-1,3-glucan binding protein (LGBP) of kuruma shrimp Marsupenaeus japonicus.

    PubMed

    Lin, Yong-Chin; Vaseeharan, Baskaralingam; Chen, Jiann-Chu

    2008-01-01

    A lipopolysaccharide (LPS) and beta-1,3-glucan binding protein (LGBP) gene was cloned from hemocytes of kuruma shrimp Marsupenaeus japonicus by reverse-transcription polymerase chain reaction (RT-PCR), cloning and sequencing of overlapping PCR, and rapid amplification of cDNA ends (RACE) method. The open reading frame (ORF) of M. japonicus LGBP is 1062 bp and encodes a 354 amino acid (aa) sequence with a 23 aa signal peptide. The calculated molecular mass of the mature protein (331 aa) is 40.15 kDa with an estimated pI of 4.78. The M. japonicus LGBP sequence contains (1) two putative N-linked glycosylation sites, (2) two putative integrin-binding motifs, (3) a kinase C phosphorylation site (KCPS), (4) a glucanase motif (GM), and (5) two potential polysaccharide recognition motifs (polysaccharide binding motif (PsBM) and beta-glucan recognition motif (GRM)), and with features of tryptophan-rich, slight homology to lysozyme, and slight homology to lectin. A sequence comparison showed that the deduced amino acids of M. japonicus LGBP has an overall high similarity to penaeid LGBP and betaGBP (85.6-89.9%), lobster Homarus gammarus betaGBP (77.0%), and crayfish Pacifastacius leniusculus LGBP (67.8%). The phylogenetic analysis revealed that M. japonicus LGBP grouped together with other crustacean LGBP and betaGBP, and was close to termite GNBP, but was far way from moth betaGBP, betaGRP, fly GNBP, and mosquito betaGRP. The LGBP of M. japonicus was strongly expressed in hemocytes. The LGBP mRNA transcript in hemocytes of M. japonicus was significantly upregulated 12-48 h after a LPS injection, indicating activation of the innate immune system through the binding of the LGBP and LPS complex. PMID:18572243

  4. G Protein-Coupled Receptor and RhoA-Stimulated Transcriptional Responses: Links to Inflammation, Differentiation, and Cell Proliferation.

    PubMed

    Yu, Olivia M; Brown, Joan Heller

    2015-07-01

    The low molecular weight G protein RhoA (rat sarcoma virus homolog family member A) serves as a node for transducing signals through G protein-coupled receptors (GPCRs). Activation of RhoA occurs through coupling of G proteins, most prominently, G12/13, to Rho guanine nucleotide exchange factors. The GPCR ligands that are most efficacious for RhoA activation include thrombin, lysophosphatidic acid, sphingosine-1-phosphate, and thromboxane A2. These ligands also stimulate proliferation, differentiation, and inflammation in a variety of cell and tissues types. The molecular events underlying these responses are the activation of transcription factors, transcriptional coactivators, and downstream gene programs. This review describes the pathways leading from GPCRs and RhoA to the regulation of activator protein-1, NFκB (nuclear factor κ-light-chain-enhancer of activated B cells), myocardin-related transcription factor A, and Yes-associated protein. We also focus on the importance of two prominent downstream transcriptional gene targets, the inflammatory mediator cyclooxygenase 2, and the matricellular protein cysteine-rich angiogenic inducer 61 (CCN1). Finally, we describe the importance of GPCR-induced activation of these pathways in the pathophysiology of cancer, fibrosis, and cardiovascular disease.

  5. The requirement for fibroblasts in angiogenesis: fibroblast-derived matrix proteins are essential for endothelial cell lumen formation

    PubMed Central

    Newman, Andrew C.; Nakatsu, Martin N.; Chou, Wayne; Gershon, Paul D.; Hughes, Christopher C. W.

    2011-01-01

    A role for fibroblasts in physiological and pathological angiogenesis is now well recognized; however, the precise mechanisms underlying their action have not been determined. Using an in vitro angiogenesis model in combination with a candidate gene approach, column chromatography, and mass spectrometry, we identify two classes of fibroblast-derived factors—one that supports vessel sprouting but not lumen formation, and one that promotes lumen formation. In the absence of fibroblasts a combination of angiopoietin-1, angiogenin, hepatocyte growth factor, transforming growth factor-α, and tumor necrosis factor drives robust endothelial cell (EC) sprouting; however, lumens fail to form. Subsequent addition of fibroblast-conditioned medium restores lumenogenesis. Using small interfering RNA–mediated knockdown, we show that five genes expressed in fibroblasts—collagen I, procollagen C endopeptidase enhancer 1, secreted protein acidic and rich in cysteine, transforming growth factor-β–induced protein ig-h3, and insulin growth factor–binding protein 7—are necessary for lumen formation. Moreover, lumen formation can be rescued by addition of purified protein to knockdown cultures. Finally, using rheology, we demonstrate that the presence of these matricellular proteins results in significantly stiffer gels, which correlates with enhanced lumen formation. These findings highlight the critical role that fibroblast-derived extracellular matrix components play in EC lumen formation and provide potential insight into the role of fibroblasts in the tumor microenvironment. PMID:21865599

  6. Modulation of endothelial cell adhesion to synthetic vascular grafts using biotinylated fibronectin in a dual ligand protein system

    NASA Astrophysics Data System (ADS)

    Anamelechi, Charles Chibuzor

    of surface immobilized protein and Trypsin/EDTA concentration. SPR results showed statistical differences in alpha5beta1 and alphanubeta3 integrin binding to RGD cell binding motifs introduced by bFN(9) and RGD-SA. Increase in binding specificity through these integrins lead to rapid cell binding and retention on Teflon-AF surfaces adsorbed with this protein formulation. This system appears to be the nexus at which the DL has proven its value. These results could have broader implications in augmenting EC attachment to SVG prior to implantation.

  7. The role of the CCN family of proteins in female reproduction.

    PubMed

    Winterhager, Elke; Gellhaus, Alexandra

    2014-06-01

    The CCN family of proteins consists of six high homologous matricellular proteins which act predominantly by binding to heparin sulphate proteoglycan and a variety of integrins. Interestingly, CCN proteins are regulated by ovarian steroid hormones and are able to adapt to changes in oxygen concentration, which is a necessary condition for successful implantation. CCN1 is involved in processes of angiogenesis within reproductive systems, thereby potentially contributing to diseases such as endometriosis and disturbed angiogenesis in the placenta and fetus. In the ovary, CCN2 is the key factor for follicular development, ovulation and corpora luteal luteolysis, and its deletion leads to fertility defects. CCN1, CCN2 and CCN3 seem to be regulators for human trophoblast proliferation and migration, but with CCN2 acting as a counterweight. Alterations in the expression of these three proteins could contribute to the shallow invasion properties observed in preeclampsia. Little is known about the role of CCN4-6 in the reproductive organs. The ability of CCN1, CCN2 and CCN3 to interact with numerous receptors enables them to adapt their biological function rapidly to the continuous remodelling of the reproductive organs and in the development of the placenta. The CCN proteins mediate their specific cell physiological function through the receptor type of their binding partner followed by a defined signalling cascade. Because of their partly overlapping expression patterns, they could act in a concert synergistically or in an opposite way within the reproductive organs. Imbalances in their expression levels are correlated to different human reproductive diseases, such as endometriosis and preeclampsia.

  8. Use of protein-engineered fabrics to identify design rules for integrin ligand clustering in biomaterials.

    PubMed

    Benitez, Patrick L; Mascharak, Shamik; Proctor, Amy C; Heilshorn, Sarah C

    2016-01-01

    While ligand clustering is known to enhance integrin activation, this insight has been difficult to apply to the design of implantable biomaterials because the local and global ligand densities that enable clustering-enhanced integrin signaling were unpredictable. Here, two general design principles for biomaterial ligand clustering are elucidated. First, clustering ligands enhances integrin-dependent signals when the global ligand density, i.e., the ligand density across the cellular length scale, is near the ligand's effective dissociation constant (KD,eff). Second, clustering ligands enhances integrin activation when the local ligand density, i.e., the ligand density across the length scale of individual focal adhesions, is less than an overcrowding threshold. To identify these principles, we fabricated a series of elastin-like, electrospun fabrics with independent control over the local (0 to 122 000 ligands μm(-2)) and global (0 to 71 000 ligand μm(-2)) densities of an arginine-glycine-aspartate (RGD) ligand. Antibody blocking studies confirmed that human umbilical vein endothelial cell adhesion to these protein-engineered biomaterials was primarily due to αVβ3 integrin binding. Clustering ligands enhanced cell proliferation, focal adhesion number, and focal adhesion kinase expression near the ligand's KD,eff of 12 000 RGD μm(-2). Near this global ligand density, cells on ligand-clustered fabrics behaved similarly to cells grown on fabrics with significantly larger global ligand densities but without clustering. However, this enhanced ligand-clustering effect was not observed above a threshold cut-off concentration. At a local ligand density of 122 000 RGD μm(-2), cell division, focal adhesion number, and focal adhesion kinase expression were significantly reduced relative to fabrics with identical global ligand density and lesser local ligand densities. Thus, when clustering results in overcrowding of ligands, integrin receptors are no longer

  9. Secreted protein acidic and rich in cysteine is a matrix scavenger chaperone.

    PubMed

    Chlenski, Alexandre; Guerrero, Lisa J; Salwen, Helen R; Yang, Qiwei; Tian, Yufeng; Morales La Madrid, Andres; Mirzoeva, Salida; Bouyer, Patrice G; Xu, David; Walker, Matthew; Cohn, Susan L

    2011-01-01

    Secreted Protein Acidic and Rich in Cysteine (SPARC) is one of the major non-structural proteins of the extracellular matrix (ECM) in remodeling tissues. The functional significance of SPARC is emphasized by its origin in the first multicellular organisms and its high degree of evolutionary conservation. Although SPARC has been shown to act as a critical modulator of ECM remodeling with profound effects on tissue physiology and architecture, no plausible molecular mechanism of its action has been proposed. In the present study, we demonstrate that SPARC mediates the disassembly and degradation of ECM networks by functioning as a matricellular chaperone. While it has low affinity to its targets inside the cells where the Ca(2+) concentrations are low, high extracellular concentrations of Ca(2+) activate binding to multiple ECM proteins, including collagens. We demonstrated that in vitro, this leads to the inhibition of collagen I fibrillogenesis and disassembly of pre-formed collagen I fibrils by SPARC at high Ca(2+) concentrations. In cell culture, exogenous SPARC was internalized by the fibroblast cells in a time- and concentration-dependent manner. Pulse-chase assay further revealed that internalized SPARC is quickly released outside the cell, demonstrating that SPARC shuttles between the cell and ECM. Fluorescently labeled collagen I, fibronectin, vitronectin, and laminin were co-internalized with SPARC by fibroblasts, and semi-quantitative Western blot showed that SPARC mediates internalization of collagen I. Using a novel 3-dimensional model of fluorescent ECM networks pre-deposited by live fibroblasts, we demonstrated that degradation of ECM depends on the chaperone activity of SPARC. These results indicate that SPARC may represent a new class of scavenger chaperones, which mediate ECM degradation, remodeling and repair by disassembling ECM networks and shuttling ECM proteins into the cell. Further understanding of this mechanism may provide insight into the

  10. Elevated cysteine-rich protein 61 (CCN1) promotes skin aging via upregulation of IL-1β in chronically sun-exposed human skin.

    PubMed

    Qin, Zhaoping; Okubo, Toru; Voorhees, John J; Fisher, Gary J; Quan, Taihao

    2014-02-01

    Chronic exposure of human skin to solar ultraviolet (UV) irradiation causes premature skin aging, which is characterized by reduced type I collagen production and increased fragmentation of the dermal collagenous extracellular matrix. This imbalance of collagen homeostasis is mediated, in part, by elevated expression of the matricellular protein cysteine-rich protein 61 (CCN1), in dermal fibroblasts, the primary collagen producing cell type in human skin. Here, we report that the actions of CCN1 are mediated by induction of interleukin 1β (IL-1β). CCN1 and IL-1β are strikingly induced by acute UV irradiation, and constitutively elevated in sun-exposed prematurely aged human skin. Elevated CCN1 rapidly induces IL-1β, inhibits type I collagen production, and upregulates matrix metalloproteinase-1, which degrades collagen fibrils. Blockade of IL-1β actions by IL-1 receptor antagonist largely prevents the deleterious effects of CCN1 on collagen homeostasis. Furthermore, knockdown of CCN1 significantly reduces induction of IL-1β by UV irradiation, and thereby partially prevents collagen loss. These data demonstrate that elevated CCN1promotes inflammaging and collagen loss via induction of IL-1β and thereby contributes to the pathophysiology of premature aging in chronically sun-exposed human skin.

  11. Pharmacological Characterization of the αvβ6 Integrin Binding and Internalization Kinetics of the Foot-and-Mouth Disease Virus Derived Peptide A20FMDV2.

    PubMed

    Slack, Robert J; Hafeji, Maryam; Rogers, Rebecca; Ludbrook, Steve B; Marshall, John F; Flint, David J; Pyne, Susan; Denyer, Jane C

    2016-01-01

    A20FMDV2 is a peptide derived from the foot-and-mouth disease virus with a high affinity and selectivity for the alpha-v beta-6 (αvβ6) arginyl-glycinyl-aspartic acid (RGD)-binding integrin. It has been shown to be an informative tool ligand in pre-clinical imaging studies for selective labelling of the αvβ6 integrin in a number of disease models. In a radioligand binding assay using a radiolabelled form of the peptide ([3H]A20FMDV2), its high affinity (K(D): 0.22 nmol/l) and selectivity (at least 85-fold) for αvβ6 over the other members of the RGD integrin family was confirmed. [3H]A20FMDV2 αvβ6 binding could be fully reversed only in the presence of EDTA, whereas a partial reversal was observed in the presence of excess concentrations of an RGD-mimetic small molecule (SC-68448) or unlabelled A20FMDV2. Using flow cytometry on bronchial epithelial cells, the ligand-induced internalization of αvβ6 by A20FMDV2 and latency-associated peptide-1 was shown to be fast (t(1/2): 1.5 and 3.1 min, respectively), concentration-dependent (EC50: values 1.1 and 3.6 nmol/l, respectively) and was followed by a moderately slow return of integrin to the surface. The results of the radioligand binding studies suggest that the binding of A20FMDV2 to the RGD-binding site on αvβ6 is required to maintain its engagement with the hypothesised A20FMDV2 synergy site on the integrin. In addition, there is evidence from flow cytometric studies that the RGD-ligand engagement of αvβ6 post-internalization plays a role in delaying recycling of the integrin to the cell surface. This mechanism may act as a homeostatic control of membrane αvβ6 following RGD ligand engagement. PMID:26734728

  12. Slow binding kinetics of secreted protein, acidic, rich in cysteine-VEGF interaction limit VEGF activation of VEGF receptor 2 and attenuate angiogenesis.

    PubMed

    Cydzik, Marzena; Abdul-Wahid, Aws; Park, Soyeon; Bourdeau, Annie; Bowden, Katherine; Prodeus, Aaron; Kollara, Alexandra; Brown, Theodore J; Ringuette, Maurice J; Gariépy, Jean

    2015-08-01

    VEGF-A (VEGF) drives angiogenesis through activation of downstream effectors to promote endothelial cell proliferation and migration. Although VEGF binds both VEGF receptor 1 (R1) and receptor 2 (R2), its proangiogenic effects are attributed to R2. Secreted protein, acidic, rich in cysteine (SPARC) is a matricellular glycoprotein thought to inhibit angiogenesis by preventing VEGF from activating R1, but not R2. Because R2 rather than R1 mediates proangiogenic activities of VEGF, the role of human SPARC in angiogenesis was reevaluated. We confirm that association of SPARC with VEGF inhibits VEGF-induced HUVEC adherence, motility, and proliferation in vitro and blocks VEGF-induced blood vessel formation ex vivo. SPARC decreases VEGF-induced phosphorylation of R2 and downstream effectors ERK, Akt, and p38 MAPK as shown by Western blot and/or phosphoflow analysis. Surface plasmon resonance indicates that SPARC binds slowly to VEGF (0.865 ± 0.02 × 10(4) M(-1) s(-1)) with a Kd of 150 nM, forming a stable complex that dissociates slowly (1.26 ± 0.003 × 10(-3) s(-1)). Only domain III of SPARC binds VEGF, exhibiting a 15-fold higher affinity than full-length SPARC. These findings support a model whereby SPARC regulates angiogenesis by sequestering VEGF, thus restricting the activation of R2 and the subsequent activation of downstream targets critical for endothelial cell functions.

  13. Galectin-8: a matricellular lectin with key roles in angiogenesis.

    PubMed

    Troncoso, María F; Ferragut, Fátima; Bacigalupo, María L; Cárdenas Delgado, Víctor M; Nugnes, Lorena G; Gentilini, Lucas; Laderach, Diego; Wolfenstein-Todel, Carlota; Compagno, Daniel; Rabinovich, Gabriel A; Elola, María T

    2014-10-01

    Galectin-8 (gal-8) is a "tandem-repeat"-type galectin, containing two carbohydrate recognition domains connected by a linker peptide. gal-8 is expressed both in the cytoplasm and nucleus in vascular endothelial cells (ECs) from normal and tumor-associated blood vessels, and in lymphatic endothelial cells. Herein, we describe a novel role for gal-8 in the regulation of vascular and lymphatic angiogenesis and provide evidence of its critical implications in tumor biology. Functional assays revealed central roles for gal-8 in the control of capillary-tube formation, EC migration and in vivo angiogenesis. So far, two endothelial ligands have been described for gal-8, namely podoplanin in lymphatic vessels and CD166 (ALCAM, activated leukocyte cell adhesion molecule) in vascular ECs. Other related gal-8 functions are also summarized here, including cell adhesion and migration, which collectively demonstrate the multi-functionality of this complex lectin. Thus, gal-8 is an important component of the angiogenesis network, and an essential molecule in the extracellular matrix by providing molecular anchoring to this surrounding matrix. The implications of gal-8 in tumor angiogenesis remain to be further explored, but it is exciting to speculate that modulating gal-8-glycan interactions could be used to block lymphatic-vascular connections vital for metastasis.

  14. Protein Condensation

    NASA Astrophysics Data System (ADS)

    Gunton, James D.; Shiryayev, Andrey; Pagan, Daniel L.

    2007-09-01

    Preface; 1. Introduction; 2. Globular protein structure; 3. Experimental methods; 4. Thermodynamics and statistical mechanics; 5. Protein-protein interactions; 6. Theoretical studies of equilibrium; 7. Nucleation theory; 8. Experimental studies of nucleation; 9. Lysozyme; 10. Some other globular proteins; 11. Membrane proteins; 12. Crystallins and cataracts; 13. Sickle hemoglobin and sickle cell anemia; 14, Alzheimer's disease; Index.

  15. Protein Condensation

    NASA Astrophysics Data System (ADS)

    Gunton, James D.; Shiryayev, Andrey; Pagan, Daniel L.

    2014-07-01

    Preface; 1. Introduction; 2. Globular protein structure; 3. Experimental methods; 4. Thermodynamics and statistical mechanics; 5. Protein-protein interactions; 6. Theoretical studies of equilibrium; 7. Nucleation theory; 8. Experimental studies of nucleation; 9. Lysozyme; 10. Some other globular proteins; 11. Membrane proteins; 12. Crystallins and cataracts; 13. Sickle hemoglobin and sickle cell anemia; 14, Alzheimer's disease; Index.

  16. Thrombospondin-1 activation of signal-regulatory protein-α stimulates reactive oxygen species production and promotes renal ischemia reperfusion injury.

    PubMed

    Yao, Mingyi; Rogers, Natasha M; Csányi, Gábor; Rodriguez, Andres I; Ross, Mark A; St Croix, Claudette; Knupp, Heather; Novelli, Enrico M; Thomson, Angus W; Pagano, Patrick J; Isenberg, Jeffrey S

    2014-06-01

    Ischemia reperfusion injury (IRI) causes tissue and organ injury, in part, through alterations in tissue blood flow and the production of reactive oxygen species. The cell surface receptor signal-regulatory protein-α (SIRP-α) is expressed on inflammatory cells and suppresses phagocytosis, but the function of SIRP-α in IRI has not been determined. We reported previously that the matricellular protein thrombospondin-1 is upregulated in IRI. Here, we report a novel interaction between thrombospondin-1 and SIRP-α on nonphagocytic cells. In cell-free experiments, thrombospondin-1 bound SIRP-α. In vascular smooth muscle cells and renal tubular epithelial cells, treatment with thrombospondin-1 led to phosphorylation of SIRP-α and downstream activation of Src homology domain 2-containing phosphatase-1. Thrombospondin-1 also stimulated phosphorylation of p47(phox) (an organizer subunit for nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 1/2) and increased production of superoxide, both of which were abrogated by knockdown or antibody blockade of SIRP-α. In rodent aortic rings, treatment with thrombospondin-1 increased the production of superoxide and inhibited nitric oxide-mediated vasodilation in a SIRP-α-dependent manner. Renal IRI upregulated the thrombospondin-1-SIRP-α signaling axis and was associated with increased superoxide production and cell death. A SIRP-α antibody that blocks thrombospondin-1 activation of SIRP-α mitigated the effects of renal IRI, increasing blood flow, suppressing production of reactive oxygen species, and preserving cellular architecture. A role for CD47 in SIRP-α activation in these pathways is also described. Overall, these results suggest that thrombospondin-1 binding to SIRP-α on nonphagocytic cells activates NADPH oxidase, limits vasodilation, and promotes renal IRI.

  17. Total protein

    MedlinePlus

    ... page: //medlineplus.gov/ency/article/003483.htm Total protein To use the sharing features on this page, please enable JavaScript. The total protein test measures the total amount of two classes ...

  18. Protein Microarrays

    NASA Astrophysics Data System (ADS)

    Ricard-Blum, S.

    Proteins are key actors in the life of the cell, involved in many physiological and pathological processes. Since variations in the expression of messenger RNA are not systematically correlated with variations in the protein levels, the latter better reflect the way a cell functions. Protein microarrays thus supply complementary information to DNA chips. They are used in particular to analyse protein expression profiles, to detect proteins within complex biological media, and to study protein-protein interactions, which give information about the functions of those proteins [3-9]. They have the same advantages as DNA microarrays for high-throughput analysis, miniaturisation, and the possibility of automation. Section 18.1 gives a brief overview of proteins. Following this, Sect. 18.2 describes how protein microarrays can be made on flat supports, explaining how proteins can be produced and immobilised on a solid support, and discussing the different kinds of substrate and detection method. Section 18.3 discusses the particular format of protein microarrays in suspension. The diversity of protein microarrays and their applications are then reported in Sect. 18.4, with applications to therapeutics (protein-drug interactions) and diagnostics. The prospects for future developments of protein microarrays are then outlined in the conclusion. The bibliography provides an extensive list of reviews and detailed references for those readers who wish to go further in this area. Indeed, the aim of the present chapter is not to give an exhaustive or detailed analysis of the state of the art, but rather to provide the reader with the basic elements needed to understand how proteins are designed and used.

  19. Dietary Proteins

    MedlinePlus

    ... meat, dairy products, nuts, and certain grains and beans. Proteins from meat and other animal products are complete proteins. This means they supply all of the amino acids the body can't make on its own. Most plant proteins are incomplete. You should eat different types ...

  20. Protein Structure

    ERIC Educational Resources Information Center

    Asmus, Elaine Garbarino

    2007-01-01

    Individual students model specific amino acids and then, through dehydration synthesis, a class of students models a protein. The students clearly learn amino acid structure, primary, secondary, tertiary, and quaternary structure in proteins and the nature of the bonds maintaining a protein's shape. This activity is fun, concrete, inexpensive and…

  1. An extracellular matrix, calmodulin-binding protein from Dictyostelium with EGF-like repeats that enhance cell motility.

    PubMed

    Suarez, Andres; Huber, Robert J; Myre, Michael A; O'Day, Danton H

    2011-07-01

    CyrA is a novel cysteine-rich protein with four EGFL repeats that was isolated using the calmodulin (CaM) binding overlay technique (CaMBOT), suggesting it is a CaM-binding protein (CaMBP). The full-length 63kDa cyrA is cleaved into two major C-terminal fragments, cyrA-C45 and cyrA-C40. A putative CaM-binding domain was detected and both CaM-agarose binding and CaM immunoprecipitation verified that cyrA-C45 and cyrA-C40 each bind to CaM in both a Ca(2+)-dependent and -independent manner. cyrA-C45 was present continuously throughout growth and development but was secreted at high levels during the multicellular slug stage of Dictyostelium development. At this time, cyrA localizes to the extracellular matrix (ECM). ECM purification verified the presence of cyrA-C45. An 18 amino acid peptide (DdEGFL1) from the first EGFL repeat sequence of cyrA (EGFL1) that is present in both cyrA-C45 and -C40 enhances both random cell motility and cAMP-mediated chemotaxis. Here we reveal that the dose-dependent enhancement of motility by DdEGFL1 is related to the time of cell starvation. Addition of DdEGFL1 also inhibits cyrA proteolysis. The status of cyrA as an extracellular CaMBP was further clarified by the demonstration that CaM is secreted during development. Antagonism of CaM with W7 resulted in enhanced cyrA proteolysis suggesting a functional role for extracellular CaM in protecting CaMBPs from proteolysis. cyrA is the first extracellular CaMBP identified in Dictyostelium and since it is an ECM protein with EGF-like repeats that enhance cell motility and it likely also represents the first matricellular protein identified in a lower eukaryote. PMID:21402150

  2. Transport proteins.

    PubMed

    Thatcher, Jack D

    2013-04-16

    This Teaching Resource provides and describes two animated lessons that illustrate general properties of transport proteins. The lesson called "transport protein classes" depicts major classes and subclasses of transport proteins. The "transporters, mechanism of action" lesson explains how transporters and P class ATPase (adenosine triphosphatase) pumps function. These animations serve as valuable resources for any collegiate-level course that describes these important factors. Courses that might use them include introductory biology, biochemistry, cell biology, physiology, and biophysics.

  3. Proteins wriggle.

    PubMed Central

    Cahill, Michael; Cahill, Sean; Cahill, Kevin

    2002-01-01

    We propose an algorithmic strategy for improving the efficiency of Monte Carlo searches for the low-energy states of proteins. Our strategy is motivated by a model of how proteins alter their shapes. In our model, when proteins fold under physiological conditions, their backbone dihedral angles change synchronously in groups of four or more to avoid steric clashes and respect the kinematic conservation laws. They wriggle; they do not thrash. We describe a simple algorithm that can be used to incorporate wriggling in Monte Carlo simulations of protein folding. We have tested this wriggling algorithm against a code in which the dihedral angles are varied independently (thrashing). Our standard of success is the average root-mean-square distance (rmsd) between the alpha-carbons of the folding protein and those of its native structure. After 100,000 Monte Carlo sweeps, the relative decrease in the mean rmsd, as one switches from thrashing to wriggling, rises from 11% for the protein 3LZM with 164 amino acids (aa) to 40% for the protein 1A1S with 313 aa and 47% for the protein 16PK with 415 aa. These results suggest that wriggling is useful and that its utility increases with the size of the protein. One may implement wriggling on a parallel computer or a computer farm. PMID:11964253

  4. Decreased astrocytic thrombospondin-1 secretion after chronic ammonia treatment reduces the level of synaptic proteins: in vitro and in vivo studies.

    PubMed

    Jayakumar, Arumugam R; Tong, Xiao Y; Curtis, Kevin M; Ruiz-Cordero, Roberto; Shamaladevi, Nagarajarao; Abuzamel, Missa; Johnstone, Joshua; Gaidosh, Gabriel; Rama Rao, Kakulavarapu V; Norenberg, Michael D

    2014-11-01

    Chronic hepatic encephalopathy (CHE) is a major complication in patients with severe liver disease. Elevated blood and brain ammonia levels have been implicated in its pathogenesis, and astrocytes are the principal neural cells involved in this disorder. Since defective synthesis and release of astrocytic factors have been shown to impair synaptic integrity in other neurological conditions, we examined whether thrombospondin-1 (TSP-1), an astrocytic factor involved in the maintenance of synaptic integrity, is also altered in CHE. Cultured astrocytes were exposed to ammonia (NH₄Cl, 0.5-2.5 mM) for 1-10 days, and TSP-1 content was measured in cell extracts and culture media. Astrocytes exposed to ammonia exhibited a reduction in intra- and extracellular TSP-1 levels. Exposure of cultured neurons to conditioned media from ammonia-treated astrocytes showed a decrease in synaptophysin, PSD95, and synaptotagmin levels. Conditioned media from TSP-1 over-expressing astrocytes that were treated with ammonia, when added to cultured neurons, reversed the decline in synaptic proteins. Recombinant TSP-1 similarly reversed the decrease in synaptic proteins. Metformin, an agent known to increase TSP-1 synthesis in other cell types, also reversed the ammonia-induced TSP-1 reduction. Likewise, we found a significant decline in TSP-1 level in cortical astrocytes, as well as a reduction in synaptophysin content in vivo in a rat model of CHE. These findings suggest that TSP-1 may represent an important therapeutic target for CHE. Defective release of astrocytic factors may impair synaptic integrity in chronic hepatic encephalopathy. We found a reduction in the release of the astrocytic matricellular proteins thrombospondin-1 (TSP-1) in ammonia-treated astrocytes; such reduction was associated with a decrease in synaptic proteins caused by conditioned media from ammonia-treated astrocytes. Exposure of neurons to CM from ammonia-treated astrocytes, in which TSP-1 is over

  5. Protein Crystallization

    NASA Technical Reports Server (NTRS)

    Chernov, Alexander A.

    2005-01-01

    Nucleation, growth and perfection of protein crystals will be overviewed along with crystal mechanical properties. The knowledge is based on experiments using optical and force crystals behave similar to inorganic crystals, though with a difference in orders of magnitude in growing parameters. For example, the low incorporation rate of large biomolecules requires up to 100 times larger supersaturation to grow protein, rather than inorganic crystals. Nucleation is often poorly reproducible, partly because of turbulence accompanying the mixing of precipitant with protein solution. Light scattering reveals fluctuations of molecular cluster size, its growth, surface energies and increased clustering as protein ages. Growth most often occurs layer-by-layer resulting in faceted crystals. New molecular layer on crystal face is terminated by a step where molecular incorporation occurs. Quantitative data on the incorporation rate will be discussed. Rounded crystals with molecularly disordered interfaces will be explained. Defects in crystals compromise the x-ray diffraction resolution crucially needed to find the 3D atomic structure of biomolecules. The defects are immobile so that birth defects stay forever. All lattice defects known for inorganics are revealed in protein crystals. Contribution of molecular conformations to lattice disorder is important, but not studied. This contribution may be enhanced by stress field from other defects. Homologous impurities (e.g., dimers, acetylated molecules) are trapped more willingly by a growing crystal than foreign protein impurities. The trapped impurities induce internal stress eliminated in crystals exceeding a critical size (part of mni for ferritin, lysozyme). Lesser impurities are trapped from stagnant, as compared to the flowing, solution. Freezing may induce much more defects unless quickly amorphysizing intracrystalline water.

  6. Bacteriophage protein-protein interactions.

    PubMed

    Häuser, Roman; Blasche, Sonja; Dokland, Terje; Haggård-Ljungquist, Elisabeth; von Brunn, Albrecht; Salas, Margarita; Casjens, Sherwood; Molineux, Ian; Uetz, Peter

    2012-01-01

    Bacteriophages T7, λ, P22, and P2/P4 (from Escherichia coli), as well as ϕ29 (from Bacillus subtilis), are among the best-studied bacterial viruses. This chapter summarizes published protein interaction data of intraviral protein interactions, as well as known phage-host protein interactions of these phages retrieved from the literature. We also review the published results of comprehensive protein interaction analyses of Pneumococcus phages Dp-1 and Cp-1, as well as coliphages λ and T7. For example, the ≈55 proteins encoded by the T7 genome are connected by ≈43 interactions with another ≈15 between the phage and its host. The chapter compiles published interactions for the well-studied phages λ (33 intra-phage/22 phage-host), P22 (38/9), P2/P4 (14/3), and ϕ29 (20/2). We discuss whether different interaction patterns reflect different phage lifestyles or whether they may be artifacts of sampling. Phages that infect the same host can interact with different host target proteins, as exemplified by E. coli phage λ and T7. Despite decades of intensive investigation, only a fraction of these phage interactomes are known. Technical limitations and a lack of depth in many studies explain the gaps in our knowledge. Strategies to complete current interactome maps are described. Although limited space precludes detailed overviews of phage molecular biology, this compilation will allow future studies to put interaction data into the context of phage biology. PMID:22748812

  7. Recombinant protein production technology

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Recombinant protein production is an important technology for antibody production, biochemical activity study, and structural determination during the post-genomic era. Limiting factors in recombinant protein production include low-level protein expression, protein precipitation, and loss of protein...

  8. Extracellular matrix mineralization in periodontal tissues: Noncollagenous matrix proteins, enzymes, and relationship to hypophosphatasia and X-linked hypophosphatemia.

    PubMed

    McKee, Marc D; Hoac, Betty; Addison, William N; Barros, Nilana M T; Millán, José L; Chaussain, Catherine

    2013-10-01

    As broadly demonstrated for the formation of a functional skeleton, proper mineralization of periodontal alveolar bone and teeth - where calcium phosphate crystals are deposited and grow within an extracellular matrix - is essential for dental function. Mineralization defects in tooth dentin and cementum of the periodontium invariably lead to a weak (soft or brittle) dentition in which teeth become loose and prone to infection and are lost prematurely. Mineralization of the extremities of periodontal ligament fibers (Sharpey's fibers) where they insert into tooth cementum and alveolar bone is also essential for the function of the tooth-suspensory apparatus in occlusion and mastication. Molecular determinants of mineralization in these tissues include mineral ion concentrations (phosphate and calcium), pyrophosphate, small integrin-binding ligand N-linked glycoproteins and matrix vesicles. Amongst the enzymes important in regulating these mineralization determinants, two are discussed at length here, with clinical examples given, namely tissue-nonspecific alkaline phosphatase and phosphate-regulating gene with homologies to endopeptidases on the X chromosome. Inactivating mutations in these enzymes in humans and in mouse models lead to the soft bones and teeth characteristic of hypophosphatasia and X-linked hypophosphatemia, respectively, where the levels of local and systemic circulating mineralization determinants are perturbed. In X-linked hypophosphatemia, in addition to renal phosphate wasting causing low circulating phosphate levels, phosphorylated mineralization-regulating small integrin-binding ligand N-linked glycoproteins, such as matrix extracellular phosphoglycoprotein and osteopontin, and the phosphorylated peptides proteolytically released from them, such as the acidic serine- and aspartate-rich-motif peptide, may accumulate locally to impair mineralization in this disease.

  9. Smooth Muscle Stiffness Sensitivity is Driven by Soluble and Insoluble ECM Chemistry

    PubMed Central

    Herrick, William G.; Rattan, Shruti; Nguyen, Thuy V.; Grunwald, Michael S.; Barney, Christopher W.; Crosby, Alfred J.; Peyton, Shelly R.

    2015-01-01

    Smooth muscle cell (SMC) invasion into plaques and subsequent proliferation is a major factor in the progression of atherosclerosis. During disease progression, SMCs experience major changes in their microenvironment, such as what integrin-binding sites are exposed, the portfolio of soluble factors available, and the elasticity and modulus of the surrounding vessel wall. We have developed a hydrogel biomaterial platform to examine the combined effect of these changes on SMC phenotype. We were particularly interested in how the chemical microenvironment affected the ability of SMCs to sense and respond to modulus. To our surprise, we observed that integrin binding and soluble factors are major drivers of several critical SMC behaviors, such as motility, proliferation, invasion, and differentiation marker expression, and these factors modulated the effect of stiffness on proliferation and migration. Overall, modulus only modestly affected behaviors other than proliferation, relative to integrin binding and soluble factors. Surprisingly, pathological behaviors (proliferation, motility) are not inversely related to SMC marker expression, in direct conflict with previous studies on substrates coupled with single extracellular matrix (ECM) proteins. A high-throughput bead-based ELISA approach and inhibitor studies revealed that differentiation marker expression is mediated chiefly via focal adhesion kinase (FAK) signaling, and we propose that integrin binding and FAK drive the transition from a migratory to a proliferative phenotype. We emphasize the importance of increasing the complexity of in vitro testing platforms to capture these subtleties in cell phenotypes and signaling, in order to better recapitulate important features of in vivo disease and elucidate potential context-dependent therapeutic targets. PMID:26495043

  10. Protein electrophoresis - serum

    MedlinePlus

    ... of protein and fat, called lipoproteins (such as LDL cholesterol). ... globulin proteins may indicate: Abnormally low level of LDL cholesterol Malnutrition Increased gamma globulin proteins may indicate: Bone ...

  11. The C-terminus of the {gamma}2 chain but not of the {beta}3 chain of laminin-332 is indirectly but indispensably necessary for integrin-mediated cell reactions

    SciTech Connect

    Navdaev, Alexei; Heitmann, Vanessa; Santana Evangelista, Karla de; Moergelin, Matthias; Wegener, Joachim; Eble, Johannes A.

    2008-02-01

    Using a recombinant mini-laminin-332, we showed that truncation of the three C-terminal amino acids of the {gamma}2 chain, but not of the C-terminal amino acid of the {beta}3 chain, completely abolished {alpha}3{beta}1 integrin binding and its cellular functions, such as attachment and spreading. However, a synthetic peptide mimicking the {gamma}2 chain C-terminus did not interfere with {alpha}3{beta}1 integrin binding or cell adhesion and spreading on laminin-332 as measured by protein interaction assays and electric cell-substrate impedance sensing. Nor was the soluble peptide able to restore the loss of integrin-mediated cell adhesiveness to mini-laminin-332 after deletion of the {gamma}2 chain C-terminus. These findings spoke against the hypothesis that the {gamma}2 chain C-terminus of laminin-332 is a part of the {alpha}3{beta}1 integrin interaction site. In addition, structural studies with electron microscopy showed that truncation of the {gamma}2 chain C-terminus opened up the compact supradomain structure of LG1-3 domains. Thus, by inducing or stabilizing an integrin binding-competent conformation or array of the LG1-3 domains, the {gamma}2 chain C-terminus plays an indirect but essential role in laminin-332 recognition by {alpha}3{beta}1 integrin and, hence, its cellular functions.

  12. Extracellular-matrix-based and Arg-Gly-Asp-modified photopolymerizing hydrogels for cartilage tissue engineering.

    PubMed

    Kim, Hwan D; Heo, Jiseung; Hwang, Yongsung; Kwak, Seon-Yeong; Park, Ok Kyu; Kim, Hyunbum; Varghese, Shyni; Hwang, Nathaniel S

    2015-02-01

    Articular cartilage damage is a persistent and increasing problem with the aging population. Strategies to achieve complete repair or functional restoration remain a challenge. Photopolymerizing-based hydrogels have long received an attention in the cartilage tissue engineering, due to their unique bioactivities, flexible method of synthesis, range of constituents, and desirable physical characteristics. In the present study, we have introduced unique bioactivity within the photopolymerizing-based hydrogels by copolymerizing polyethylene glycol (PEG) macromers with methacrylated extracellular matrix (ECM) molecules (hyaluronic acid and chondroitin sulfate [CS]) and integrin binding peptides (RGD peptide). Results indicate that cellular morphology, as observed by the actin cytoskeleton structures, was strongly dependent on the type of ECM component as well as the presence of integrin binding moieties. Further, CS-based hydrogel with integrin binding RGD moieties increased the lubricin (or known as superficial zone protein [SZP]) gene expression of the encapsulated chondrocytes. Additionally, CS-based hydrogel displayed cell-responsive degradation and resulted in increased DNA, GAG, and collagen accumulation compared with other hydrogels. This study demonstrates that integrin-mediated interactions within CS microenvironment provide an optimal hydrogel scaffold for cartilage tissue engineering application.

  13. Extracellular-matrix-based and Arg-Gly-Asp-modified photopolymerizing hydrogels for cartilage tissue engineering.

    PubMed

    Kim, Hwan D; Heo, Jiseung; Hwang, Yongsung; Kwak, Seon-Yeong; Park, Ok Kyu; Kim, Hyunbum; Varghese, Shyni; Hwang, Nathaniel S

    2015-02-01

    Articular cartilage damage is a persistent and increasing problem with the aging population. Strategies to achieve complete repair or functional restoration remain a challenge. Photopolymerizing-based hydrogels have long received an attention in the cartilage tissue engineering, due to their unique bioactivities, flexible method of synthesis, range of constituents, and desirable physical characteristics. In the present study, we have introduced unique bioactivity within the photopolymerizing-based hydrogels by copolymerizing polyethylene glycol (PEG) macromers with methacrylated extracellular matrix (ECM) molecules (hyaluronic acid and chondroitin sulfate [CS]) and integrin binding peptides (RGD peptide). Results indicate that cellular morphology, as observed by the actin cytoskeleton structures, was strongly dependent on the type of ECM component as well as the presence of integrin binding moieties. Further, CS-based hydrogel with integrin binding RGD moieties increased the lubricin (or known as superficial zone protein [SZP]) gene expression of the encapsulated chondrocytes. Additionally, CS-based hydrogel displayed cell-responsive degradation and resulted in increased DNA, GAG, and collagen accumulation compared with other hydrogels. This study demonstrates that integrin-mediated interactions within CS microenvironment provide an optimal hydrogel scaffold for cartilage tissue engineering application. PMID:25266634

  14. Protein sulfhydration.

    PubMed

    Paul, Bindu D; Snyder, Solomon H

    2015-01-01

    Hydrogen sulfide (H2S) is one of the gasotransmitters that modulates various biological processes and participates in multiple signaling pathways. H2S signals by a process termed sulfhydration. Sulfhydration has recently been recognized as a posttranslational modification similar to nitrosylation. Sulfhydration occurs at reactive cysteine residues in proteins and results in the conversion of an -SH group of cysteine to an -SSH or a persulfide group. Sulfhydration is highly prevalent in vivo, and aberrant sulfhydration patterns have been observed under several pathological conditions ranging from heart disease to neurodegenerative diseases such as Parkinson's disease. The biotin switch assay, originally developed to detect nitrosylation, has been modified to detect sulfhydration. In this chapter, we discuss the physiological roles of sulfhydration and the methodologies used to detect this modification.

  15. Fusion-protein-assisted protein crystallization.

    PubMed

    Kobe, Bostjan; Ve, Thomas; Williams, Simon J

    2015-07-01

    Fusion proteins can be used directly in protein crystallization to assist crystallization in at least two different ways. In one approach, the `heterologous fusion-protein approach', the fusion partner can provide additional surface area to promote crystal contact formation. In another approach, the `fusion of interacting proteins approach', protein assemblies can be stabilized by covalently linking the interacting partners. The linker connecting the proteins plays different roles in the two applications: in the first approach a rigid linker is required to reduce conformational heterogeneity; in the second, conversely, a flexible linker is required that allows the native interaction between the fused proteins. The two approaches can also be combined. The recent applications of fusion-protein technology in protein crystallization from the work of our own and other laboratories are briefly reviewed.

  16. EDITORIAL: Precision proteins Precision proteins

    NASA Astrophysics Data System (ADS)

    Demming, Anna

    2010-06-01

    Since the birth of modern day medicine, during the times of Hippocrates in ancient Greece, the profession has developed from the rudimentary classification of disease into a rigorous science with an inspiring capability to treat and cure. Scientific methodology has distilled clinical diagnostic tools from the early arts of prognosis, which used to rely as much on revelation and prophecy, as intuition and judgement [1]. Over the past decade, research into the interactions between proteins and nanosystems has provided some ingenious and apt techniques for delving into the intricacies of anatomical systems. In vivo biosensing has emerged as a vibrant field of research, as much of medical diagnosis relies on the detection of substances or an imbalance in the chemicals in the body. The inherent properties of nanoscale structures, such as cantilevers, make them well suited to biosensing applications that demand the detection of molecules at very low concentrations. Measurable deflections in cantilevers functionalised with antibodies provide quantitative indicators of the presence of specific antigens when the two react. Such developments have roused mounting interest in the interactions of proteins with nanostructures, such as carbon nanotubes [3], which have demonstrated great potential as generic biomarkers. Plasmonic properties are also being exploited in sensing applications, such as the molecular sentinel recently devised by researchers in the US. The device uses the plasmonic properties of a silver nanoparticle linked to a Raman labelled hairpin DNA probe to signal changes in the probe geometry resulting from interactions with substances in the environment. Success stories so far include the detection of two specific genes associated with breast cancer [4]. A greater understanding of how RNA interference regulates gene expression has highlighted the potential of using this natural process as another agent for combating disease in personalized medicine. However, the

  17. EDITORIAL: Precision proteins Precision proteins

    NASA Astrophysics Data System (ADS)

    Demming, Anna

    2010-06-01

    Since the birth of modern day medicine, during the times of Hippocrates in ancient Greece, the profession has developed from the rudimentary classification of disease into a rigorous science with an inspiring capability to treat and cure. Scientific methodology has distilled clinical diagnostic tools from the early arts of prognosis, which used to rely as much on revelation and prophecy, as intuition and judgement [1]. Over the past decade, research into the interactions between proteins and nanosystems has provided some ingenious and apt techniques for delving into the intricacies of anatomical systems. In vivo biosensing has emerged as a vibrant field of research, as much of medical diagnosis relies on the detection of substances or an imbalance in the chemicals in the body. The inherent properties of nanoscale structures, such as cantilevers, make them well suited to biosensing applications that demand the detection of molecules at very low concentrations. Measurable deflections in cantilevers functionalised with antibodies provide quantitative indicators of the presence of specific antigens when the two react. Such developments have roused mounting interest in the interactions of proteins with nanostructures, such as carbon nanotubes [3], which have demonstrated great potential as generic biomarkers. Plasmonic properties are also being exploited in sensing applications, such as the molecular sentinel recently devised by researchers in the US. The device uses the plasmonic properties of a silver nanoparticle linked to a Raman labelled hairpin DNA probe to signal changes in the probe geometry resulting from interactions with substances in the environment. Success stories so far include the detection of two specific genes associated with breast cancer [4]. A greater understanding of how RNA interference regulates gene expression has highlighted the potential of using this natural process as another agent for combating disease in personalized medicine. However, the

  18. Protein Crystal Based Nanomaterials

    NASA Technical Reports Server (NTRS)

    Bell, Jeffrey A.; VanRoey, Patrick

    2001-01-01

    This is the final report on a NASA Grant. It concerns a description of work done, which includes: (1) Protein crystals cross-linked to form fibers; (2) Engineering of protein to favor crystallization; (3) Better knowledge-based potentials for protein-protein contacts; (4) Simulation of protein crystallization.

  19. Shotgun protein sequencing.

    SciTech Connect

    Faulon, Jean-Loup Michel; Heffelfinger, Grant S.

    2009-06-01

    A novel experimental and computational technique based on multiple enzymatic digestion of a protein or protein mixture that reconstructs protein sequences from sequences of overlapping peptides is described in this SAND report. This approach, analogous to shotgun sequencing of DNA, is to be used to sequence alternative spliced proteins, to identify post-translational modifications, and to sequence genetically engineered proteins.

  20. Protein immobilization strategies for protein biochips.

    PubMed

    Rusmini, Federica; Zhong, Zhiyuan; Feijen, Jan

    2007-06-01

    In the past few years, protein biochips have emerged as promising proteomic and diagnostic tools for obtaining information about protein functions and interactions. Important technological innovations have been made. However, considerable development is still required, especially regarding protein immobilization, in order to fully realize the potential of protein biochips. In fact, protein immobilization is the key to the success of microarray technology. Proteins need to be immobilized onto surfaces with high density in order to allow the usage of small amount of sample solution. Nonspecific protein adsorption needs to be avoided or at least minimized in order to improve detection performances. Moreover, full retention of protein conformation and activity is a challenging task to be accomplished. Although a large number of review papers on protein biochips have been published in recent years, few have focused on protein immobilization technology. In this review, current protein immobilization strategies, including physical, covalent, and bioaffinity immobilization for the fabrication of protein biochips, are described. Particular consideration has been given to oriented immobilization, also referred to as site-specific immobilization, which is believed will improve homogeneous surface covering and accessibility of the active site.

  1. Protein-losing enteropathy

    MedlinePlus

    ... this page: //medlineplus.gov/ency/article/007338.htm Protein-losing enteropathy To use the sharing features on this page, please enable JavaScript. Protein-losing enteropathy is an abnormal loss of protein ...

  2. Protein in diet

    MedlinePlus

    ... basic structure of protein is a chain of amino acids. You need protein in your diet to help ... Protein foods are broken down into parts called amino acids during digestion. The human body needs a number ...

  3. Nanotechnologies in protein microarrays.

    PubMed

    Krizkova, Sona; Heger, Zbynek; Zalewska, Marta; Moulick, Amitava; Adam, Vojtech; Kizek, Rene

    2015-01-01

    Protein microarray technology became an important research tool for study and detection of proteins, protein-protein interactions and a number of other applications. The utilization of nanoparticle-based materials and nanotechnology-based techniques for immobilization allows us not only to extend the surface for biomolecule immobilization resulting in enhanced substrate binding properties, decreased background signals and enhanced reporter systems for more sensitive assays. Generally in contemporarily developed microarray systems, multiple nanotechnology-based techniques are combined. In this review, applications of nanoparticles and nanotechnologies in creating protein microarrays, proteins immobilization and detection are summarized. We anticipate that advanced nanotechnologies can be exploited to expand promising fields of proteins identification, monitoring of protein-protein or drug-protein interactions, or proteins structures. PMID:26039143

  4. Protein domain architectures.

    PubMed

    Mulder, Nicola J

    2010-01-01

    Proteins are composed of functional units, or domains, that can be found alone or in combination with other domains. Analysis of protein domain architectures and the movement of protein domains within and across different genomes provide clues about the evolution of protein function. The classification of proteins into families and domains is provided through publicly available tools and databases that use known protein domains to predict other members in new proteins sequences. Currently at least 80% of the main protein sequence databases can be classified using these tools, thus providing a large data set to work from for analyzing protein domain architectures. Each of the protein domain databases provide intuitive web interfaces for viewing and analyzing their domain classifications and provide their data freely for downloading. Some of the main protein family and domain databases are described here, along with their Web-based tools for analyzing domain architectures.

  5. PREFACE: Protein protein interactions: principles and predictions

    NASA Astrophysics Data System (ADS)

    Nussinov, Ruth; Tsai, Chung-Jung

    2005-06-01

    Proteins are the `workhorses' of the cell. Their roles span functions as diverse as being molecular machines and signalling. They carry out catalytic reactions, transport, form viral capsids, traverse membranes and form regulated channels, transmit information from DNA to RNA, making possible the synthesis of new proteins, and they are responsible for the degradation of unnecessary proteins and nucleic acids. They are the vehicles of the immune response and are responsible for viral entry into the cell. Given their importance, considerable effort has been centered on the prediction of protein function. A prime way to do this is through identification of binding partners. If the function of at least one of the components with which the protein interacts is known, that should let us assign its function(s) and the pathway(s) in which it plays a role. This holds since the vast majority of their chores in the living cell involve protein-protein interactions. Hence, through the intricate network of these interactions we can map cellular pathways, their interconnectivities and their dynamic regulation. Their identification is at the heart of functional genomics; their prediction is crucial for drug discovery. Knowledge of the pathway, its topology, length, and dynamics may provide useful information for forecasting side effects. The goal of predicting protein-protein interactions is daunting. Some associations are obligatory, others are continuously forming and dissociating. In principle, from the physical standpoint, any two proteins can interact, but under what conditions and at which strength? The principles of protein-protein interactions are general: the non-covalent interactions of two proteins are largely the outcome of the hydrophobic effect, which drives the interactions. In addition, hydrogen bonds and electrostatic interactions play important roles. Thus, many of the interactions observed in vitro are the outcome of experimental overexpression. Protein disorder

  6. Protein sequence comparison and protein evolution

    SciTech Connect

    Pearson, W.R.

    1995-12-31

    This tutorial was one of eight tutorials selected to be presented at the Third International Conference on Intelligent Systems for Molecular Biology which was held in the United Kingdom from July 16 to 19, 1995. This tutorial examines how the information conserved during the evolution of a protein molecule can be used to infer reliably homology, and thus a shared proteinfold and possibly a shared active site or function. The authors start by reviewing a geological/evolutionary time scale. Next they look at the evolution of several protein families. During the tutorial, these families will be used to demonstrate that homologous protein ancestry can be inferred with confidence. They also examine different modes of protein evolution and consider some hypotheses that have been presented to explain the very earliest events in protein evolution. The next part of the tutorial will examine the technical aspects of protein sequence comparison. Both optimal and heuristic algorithms and their associated parameters that are used to characterize protein sequence similarities are discussed. Perhaps more importantly, they survey the statistics of local similarity scores, and how these statistics can both be used to improve the selectivity of a search and to evaluate the significance of a match. They them examine distantly related members of three protein families, the serine proteases, the glutathione transferases, and the G-protein-coupled receptors (GCRs). Finally, the discuss how sequence similarity can be used to examine internal repeated or mosaic structures in proteins.

  7. Inferring Protein Associations Using Protein Pulldown Assays

    SciTech Connect

    Sharp, Julia L.; Anderson, Kevin K.; Daly, Don S.; Auberry, Deanna L.; Borkowski, John J.; Cannon, William R.

    2007-02-01

    Background: One method to infer protein-protein associations is through a “bait-prey pulldown” assay using a protein affinity agent and an LC-MS (liquid chromatography-mass spectrometry)-based protein identification method. False positive and negative protein identifications are not uncommon, however, leading to incorrect inferences. Methods: A pulldown experiment generates a protein association matrix wherein each column represents a sample from one bait protein, each row represents one prey protein and each cell contains a presence/absence association indicator. Our method evaluates the presence/absence pattern across a prey protein (row) with a Likelihood Ratio Test (LRT), computing its p-value with simulated LRT test statistic distributions after a check with simulated binomial random variates disqualified the large sample 2 test. A pulldown experiment often involves hundreds of tests so we apply the false discovery rate method to control the false positive rate. Based on the p-value, each prey protein is assigned a category (specific association, non-specific association, or not associated) and appraised with respect to the pulldown experiment’s goal and design. The method is illustrated using a pulldown experiment investigating the protein complexes of Shewanella oneidensis MR-1. Results: The Monte Carlo simulated LRT p-values objectively reveal specific and ubiquitous prey, as well as potential systematic errors. The example analysis shows the results to be biologically sensible and more realistic than the ad hoc screening methods previously utilized. Conclusions: The method presented appears to be informative for screening for protein-protein associations.

  8. Mirror image proteins.

    PubMed

    Zhao, Le; Lu, Wuyuan

    2014-10-01

    Proteins composed entirely of unnatural d-amino acids and the achiral amino acid glycine are mirror image forms of their native l-protein counterparts. Recent advances in chemical protein synthesis afford unique and facile synthetic access to domain-sized mirror image d-proteins, enabling protein research to be conducted through 'the looking glass' and in a way previously unattainable. d-Proteins can facilitate structure determination of their native l-forms that are difficult to crystallize (racemic X-ray crystallography); d-proteins can serve as the bait for library screening to ultimately yield pharmacologically superior d-peptide/d-protein therapeutics (mirror-image phage display); d-proteins can also be used as a powerful mechanistic tool for probing molecular events in biology. This review examines recent progress in the application of mirror image proteins to structural biology, drug discovery, and immunology.

  9. High-throughput and multiplexed protein array technology: protein-DNA and protein-protein interactions.

    PubMed

    Sakanyan, Vehary

    2005-02-01

    Miniaturized protein arrays address protein interactions with various types of molecules in a high-throughput and multiplexed fashion. This review focuses on achievements in the analysis of protein-DNA and protein-protein interactions. The technological feasibility of protein arrays depends on the different factors that enable the arrayed proteins to recognize molecular partners and on the specificity of the interactions involved. Proteome-scale studies of molecular interactions require high-throughput approaches for both the production and purification of functionally active proteins. Various solutions have been proposed to avoid non-specific protein interactions on array supports and to monitor low-abundance molecules. The data accumulated indicate that this emerging technology is perfectly suited to resolve networks of protein interactions involved in complex physiological and pathological phenomena in different organisms and to develop sensitive tools for biomedical applications.

  10. Protein- protein interaction detection system using fluorescent protein microdomains

    DOEpatents

    Waldo, Geoffrey S.; Cabantous, Stephanie

    2010-02-23

    The invention provides a protein labeling and interaction detection system based on engineered fragments of fluorescent and chromophoric proteins that require fused interacting polypeptides to drive the association of the fragments, and further are soluble and stable, and do not change the solubility of polypeptides to which they are fused. In one embodiment, a test protein X is fused to a sixteen amino acid fragment of GFP (.beta.-strand 10, amino acids 198-214), engineered to not perturb fusion protein solubility. A second test protein Y is fused to a sixteen amino acid fragment of GFP (.beta.-strand 11, amino acids 215-230), engineered to not perturb fusion protein solubility. When X and Y interact, they bring the GFP strands into proximity, and are detected by complementation with a third GFP fragment consisting of GFP amino acids 1-198 (strands 1-9). When GFP strands 10 and 11 are held together by interaction of protein X and Y, they spontaneous association with GFP strands 1-9, resulting in structural complementation, folding, and concomitant GFP fluorescence.

  11. [Protein expression and purification].

    PubMed

    Růčková, E; Müller, P; Vojtěšek, B

    2014-01-01

    Production of recombinant proteins is essential for many applications in both basic research and also in medicine, where recombinant proteins are used as pharmaceuticals. This review summarizes procedures involved in recombinant protein expression and purification, including molecular cloning of target genes into expression vectors, selection of the appropriate expression system, and protein purification techniques. Recombinant DNA technology allows protein engineering to modify protein stability, activity and function or to facilitate protein purification by affinity tag fusions. A wide range of cloning systems enabling fast and effective design of expression vectors is currently available. A first choice of protein expression system is usually the bacteria Escherichia coli. The main advantages of this prokaryotic expression system are low cost and simplicity; on the other hand this system is often unsuitable for production of complex mammalian proteins. Protein expression mediated by eukaryotic cells (yeast, insect and mammalian cells) usually produces properly folded and posttranslationally modified proteins. How-ever, cultivation of insect and, especially, mammalian cells is time consuming and expensive. Affinity tagged recombinant proteins are purified efficiently using affinity chromatography. An affinity tag is a protein or peptide that mediates specific binding to a chromatography column, unbound proteins are removed during a washing step and pure protein is subsequently eluted. PMID:24945544

  12. Urine Protein and Urine Protein to Creatinine Ratio

    MedlinePlus

    ... limited. Home Visit Global Sites Search Help? Urine Protein and Urine Protein to Creatinine Ratio Share this page: Was this page helpful? Also known as: 24-Hour Urine Protein; Urine Total Protein; Urine Protein to Creatinine Ratio; ...

  13. Designing Fluorinated Proteins.

    PubMed

    Marsh, E N G

    2016-01-01

    As methods to incorporate noncanonical amino acid residues into proteins have become more powerful, interest in their use to modify the physical and biological properties of proteins and enzymes has increased. This chapter discusses the use of highly fluorinated analogs of hydrophobic amino acids, for example, hexafluoroleucine, in protein design. In particular, fluorinated residues have proven to be generally effective in increasing the thermodynamic stability of proteins. The chapter provides an overview of the different fluorinated amino acids that have been used in protein design and the various methods available for producing fluorinated proteins. It discusses model proteins systems into which highly fluorinated amino acids have been introduced and the reasons why fluorinated residues are generally stabilizing, with particular reference to thermodynamic and structural studies from our laboratory. Lastly, details of the methodology we have developed to measure the thermodynamic stability of oligomeric fluorinated proteins are presented, as this may be generally applicable to many proteins. PMID:27586337

  14. DNA mimicry by proteins.

    PubMed

    Dryden, D T F; Tock, M R

    2006-04-01

    It has been discovered recently, via structural and biophysical analyses, that proteins can mimic DNA structures in order to inhibit proteins that would normally bind to DNA. Mimicry of the phosphate backbone of DNA, the hydrogen-bonding properties of the nucleotide bases and the bending and twisting of the DNA double helix are all present in the mimics discovered to date. These mimics target a range of proteins and enzymes such as DNA restriction enzymes, DNA repair enzymes, DNA gyrase and nucleosomal and nucleoid-associated proteins. The unusual properties of these protein DNA mimics may provide a foundation for the design of targeted inhibitors of DNA-binding proteins. PMID:16545103

  15. Physics of protein motility and motor proteins

    NASA Astrophysics Data System (ADS)

    Kolomeisky, Anatoly B.

    2013-09-01

    Motor proteins are enzymatic molecules that transform chemical energy into mechanical motion and work. They are critically important for supporting various cellular activities and functions. In the last 15 years significant progress in understanding the functioning of motor proteins has been achieved due to revolutionary breakthroughs in single-molecule experimental techniques and strong advances in theoretical modelling. However, microscopic mechanisms of protein motility are still not well explained, and the collective efforts of many scientists are needed in order to solve these complex problems. In this special section the reader will find the latest advances on the difficult road to mapping motor proteins dynamics in various systems. Recent experimental developments have allowed researchers to monitor and to influence the activity of single motor proteins with a high spatial and temporal resolution. It has stimulated significant theoretical efforts to understand the non-equilibrium nature of protein motility phenomena. The latest results from all these advances are presented and discussed in this special section. We would like to thank the scientists from all over the world who have reported their latest research results for this special section. We are also grateful to the staff and editors of Journal of Physics: Condensed Matter for their invaluable help in handling all the administrative and refereeing activities. The field of motor proteins and protein motility is fast moving, and we hope that this collection of articles will be a useful source of information in this highly interdisciplinary area. Physics of protein motility and motor proteins contents Physics of protein motility and motor proteinsAnatoly B Kolomeisky Identification of unique interactions between the flexible linker and the RecA-like domains of DEAD-box helicase Mss116 Yuan Zhang, Mirkó Palla, Andrew Sun and Jung-Chi Liao The load dependence of the physical properties of a molecular motor

  16. Protein and protein hydrolysates in sports nutrition.

    PubMed

    van Loon, Luc J C; Kies, Arie K; Saris, Wim H M

    2007-08-01

    With the increasing knowledge about the role of nutrition in increasing exercise performance, it has become clear over the last 2 decades that amino acids, protein, and protein hydrolysates can play an important role. Most of the attention has been focused on their effects at a muscular level. As these nutrients are ingested, however, it also means that gastrointestinal digestibility and absorption can modulate their efficacy significantly. Therefore, discussing the role of amino acids, protein, and protein hydrolysates in sports nutrition entails holding a discussion on all levels of the metabolic route. On May 28-29, 2007, a small group of researchers active in the field of exercise science and protein metabolism presented an overview of the different aspects of the application of protein and protein hydrolysates in sports nutrition. In addition, they were asked to share their opinions on the future progress in their fields of research. In this overview, an introduction to the workshop and a short summary of its outcome is provided.

  17. Engineering fluorescent proteins.

    PubMed

    Miyawaki, Atsushi; Nagai, Takeharu; Mizuno, Hideaki

    2005-01-01

    Green fluorescent protein from the jellyfish Aequorea victora (GFP) and GFP-like proteins from Anthozoa species encode light-absorbing chromophores intrinsically within their respective protein sequences. Recent studies have made progress in obtaining bright variants of these proteins which develop chromophores quickly and efficiently, as well as novel fluorescent proteins that photoactivate or photoconvert, i.e., become fluorescent or change colors upon illumination at specific wavelengths. Also, monomeric versions of these proteins have been engineered for fusion protein applications. Simple GFP variants and circularly permuted GFP variants have been used to develop fluorescent probes that sense physiological signals such as membrane potential and concentrations of free calcium. Further molecular characterization of the structure and maturation of these proteins is in progress, aimed at providing information for rational design of variants with desired fluorescence properties.

  18. Learning about Proteins

    MedlinePlus

    ... body, and protecting you from disease. All About Amino Acids When you eat foods that contain protein, the ... called amino (say: uh-MEE-no) acids. The amino acids then can be reused to make the proteins ...

  19. Protein S blood test

    MedlinePlus

    ... a normal substance in your body that prevents blood clotting. A blood test can be done to see ... family history of blood clots. Protein S helps control blood clotting. A lack of this protein or problem with ...

  20. Protein C blood test

    MedlinePlus

    ... a normal substance in the body that prevents blood clotting. A blood test can be done to see ... history of blood clots. Protein C helps control blood clotting. A lack of this protein or problem with ...

  1. [Protein-losing enteropathy].

    PubMed

    Amiot, A

    2015-07-01

    Protein-losing enteropathy is a rare syndrome of gastrointestinal protein loss. The primary causes can be classified into lymphatic leakage due to increased interstitial pressure and increased leakage of protein-rich fluids due to erosive or non-erosive gastrointestinal disorders. The diagnosis of protein-losing enteropathy should be considered in patients with chronic diarrhea and peripheral oedema. The diagnosis of protein-losing enteropathy is most commonly based on the determination of fecal alpha-1 antitrypsin clearance. Most protein-losing enteropathy cases are the result of either lymphatic obstruction or a variety of gastrointestinal disorders and cardiac diseases, while primary intestinal lymphangiectasia (Waldmann's disease) is less common. Treatment of protein-losing enteropathy targets the underlying disease but also includes dietary modification, such as high-protein and low-fat diet along with medium-chain triglyceride supplementation. PMID:25618488

  2. Protein and older adults.

    PubMed

    Chernoff, Ronni

    2004-12-01

    Body composition changes as people get older. One of the noteworthy alterations is the reduction in total body protein. A decrease in skeletal muscle is the most noticeable manifestation of this change but there is also a reduction in other physiologic proteins such as organ tissue, blood components, and immune bodies as well as declines in total body potassium and water. This contributes to impaired wound healing, loss of skin elasticity, and an inability to fight infection. The recommended dietary allowance (RDA) for adults for protein is 0.8 grams of protein per kilogram of body weight. Protein tissue accounts for 30% of whole-body protein turnover but that rate declines to 20% or less by age 70. The result of this phenomenon is that older adults require more protein/kilogram body weight than do younger adults. Recently, it has become clear that the requirement for exogenous protein is at least 1.0 gram/kilogram body weight. Adequate dietary intake of protein may be more difficult for older adults to obtain. Dietary animal protein is the primary source of high biological value protein, iron, vitamin B(12), folic acid, biotin and other essential nutrients. In fact, egg protein is the standard against which all other proteins are compared. Compared to other high-quality protein sources like meat, poultry and seafood, eggs are the least expensive. The importance of dietary protein cannot be underestimated in the diets of older adults; inadequate protein intake contributes to a decrease in reserve capacity, increased skin fragility, decreased immune function, poorer healing, and longer recuperation from illness.

  3. Texturized dairy proteins

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Dairy proteins are amenable to structural modifications induced by high temperature, shear and moisture; in particular, whey proteins can change conformation to new unfolded states. The change in protein state is a basis for creating new foods. The dairy products, nonfat dried milk (NDM), whey prote...

  4. Modeling Protein Self Assembly

    ERIC Educational Resources Information Center

    Baker, William P.; Jones, Carleton Buck; Hull, Elizabeth

    2004-01-01

    Understanding the structure and function of proteins is an important part of the standards-based science curriculum. Proteins serve vital roles within the cell and malfunctions in protein self assembly are implicated in degenerative diseases. Experience indicates that this topic is a difficult one for many students. We have found that the concept…

  5. Destabilized bioluminescent proteins

    DOEpatents

    Allen, Michael S.; Rakesh, Gupta; Gary, Sayler S.

    2007-07-31

    Purified nucleic acids, vectors and cells containing a gene cassette encoding at least one modified bioluminescent protein, wherein the modification includes the addition of a peptide sequence. The duration of bioluminescence emitted by the modified bioluminescent protein is shorter than the duration of bioluminescence emitted by an unmodified form of the bioluminescent protein.

  6. Modeling Protein Domain Function

    ERIC Educational Resources Information Center

    Baker, William P.; Jones, Carleton "Buck"; Hull, Elizabeth

    2007-01-01

    This simple but effective laboratory exercise helps students understand the concept of protein domain function. They use foam beads, Styrofoam craft balls, and pipe cleaners to explore how domains within protein active sites interact to form a functional protein. The activity allows students to gain content mastery and an understanding of the…

  7. CSF total protein

    MedlinePlus

    CSF total protein is a test to determine the amount of protein in your spinal fluid, also called cerebrospinal fluid (CSF). ... The normal protein range varies from lab to lab, but is typically about 15 to 60 mg/dL. Note: mg/dL = ...

  8. Protopia: a protein-protein interaction tool

    PubMed Central

    Real-Chicharro, Alejandro; Ruiz-Mostazo, Iván; Navas-Delgado, Ismael; Kerzazi, Amine; Chniber, Othmane; Sánchez-Jiménez, Francisca; Medina, Miguel Ángel; Aldana-Montes, José F

    2009-01-01

    Background Protein-protein interactions can be considered the basic skeleton for living organism self-organization and homeostasis. Impressive quantities of experimental data are being obtained and computational tools are essential to integrate and to organize this information. This paper presents Protopia, a biological tool that offers a way of searching for proteins and their interactions in different Protein Interaction Web Databases, as a part of a multidisciplinary initiative of our institution for the integration of biological data . Results The tool accesses the different Databases (at present, the free version of Transfac, DIP, Hprd, Int-Act and iHop), and results are expressed with biological protein names or databases codes and can be depicted as a vector or a matrix. They can be represented and handled interactively as an organic graph. Comparison among databases is carried out using the Uniprot codes annotated for each protein. Conclusion The tool locates and integrates the current information stored in the aforementioned databases, and redundancies among them are detected. Results are compatible with the most important network analysers, so that they can be compared and analysed by other world-wide known tools and platforms. The visualization possibilities help to attain this goal and they are especially interesting for handling multiple-step or complex networks. PMID:19828077

  9. Viral complement regulatory proteins.

    PubMed

    Rosengard, A M; Ahearn, J M

    1999-05-01

    The inactivation of complement provides cells and tissues critical protection from complement-mediated attack and decreases the associated recruitment of other inflammatory mediators. In an attempt to evade the host immune response, viruses have evolved two mechanisms to acquire complement regulatory proteins. They can directly seize the host cell complement regulators onto their outer envelope and/or they can produce their own proteins which are either secreted into the neighboring intercellular space or expressed as membrane-bound proteins on the infected host cell. The following review will concentrate on the viral homologues of the mammalian complement regulatory proteins, specifically those containing complement control protein (CCP) repeats. PMID:10408371

  10. Highly thermostable fluorescent proteins

    DOEpatents

    Bradbury, Andrew M.; Waldo, Geoffrey S.; Kiss, Csaba

    2011-03-22

    Thermostable fluorescent proteins (TSFPs), methods for generating these and other stability-enhanced proteins, polynucleotides encoding such proteins, and assays and method for using the TSFPs and TSFP-encoding nucleic acid molecules are provided. The TSFPs of the invention show extremely enhanced levels of stability and thermotolerance. In one case, for example, a TSFP of the invention is so stable it can be heated to 99.degree. C. for short periods of time without denaturing, and retains 85% of its fluorescence when heated to 80.degree. C. for several minutes. The invention also provides a method for generating stability-enhanced variants of a protein, including but not limited to fluorescent proteins.

  11. Highly thermostable fluorescent proteins

    DOEpatents

    Bradbury, Andrew M.; Waldo, Geoffrey S.; Kiss, Csaba

    2012-05-01

    Thermostable fluorescent proteins (TSFPs), methods for generating these and other stability-enhanced proteins, polynucleotides encoding such proteins, and assays and method for using the TSFPs and TSFP-encoding nucleic acid molecules are provided. The TSFPs of the invention show extremely enhanced levels of stability and thermotolerance. In one case, for example, a TSFP of the invention is so stable it can be heated to 99.degree. C. for short periods of time without denaturing, and retains 85% of its fluorescence when heated to 80.degree. C. for several minutes. The invention also provides a method for generating stability-enhanced variants of a protein, including but not limited to fluorescent proteins.

  12. Highly thermostable fluorescent proteins

    DOEpatents

    Bradbury, Andrew M.; Waldo, Geoffrey S.; Kiss, Csaba

    2011-11-29

    Thermostable fluorescent proteins (TSFPs), methods for generating these and other stability-enhanced proteins, polynucleotides encoding such proteins, and assays and method for using the TSFPs and TSFP-encoding nucleic acid molecules are provided. The TSFPs of the invention show extremely enhanced levels of stability and thermotolerance. In one case, for example, a TSFP of the invention is so stable it can be heated to 99.degree. C. for short periods of time without denaturing, and retains 85% of its fluorescence when heated to 80.degree. C. for several minutes. The invention also provides a method for generating stability-enhanced variants of a protein, including but not limited to fluorescent proteins.

  13. Selective Precipitation of Proteins.

    PubMed

    Matulis, Daumantas

    2016-01-01

    Selective precipitation of proteins can be used as a bulk method to recover the majority of proteins from a crude lysate, as a selective method to fractionate a subset of proteins from a protein solution, or as a very specific method to recover a single protein of interest from a purification step. This unit describes a number of methods suitable for selective precipitation. In each of the protocols that are outlined, the physical or chemical basis of the precipitation process, the parameters that can be varied for optimization, and the basic steps for developing an optimized precipitation are described.

  14. Protein crystallization with paper

    NASA Astrophysics Data System (ADS)

    Matsuoka, Miki; Kakinouchi, Keisuke; Adachi, Hiroaki; Maruyama, Mihoko; Sugiyama, Shigeru; Sano, Satoshi; Yoshikawa, Hiroshi Y.; Takahashi, Yoshinori; Yoshimura, Masashi; Matsumura, Hiroyoshi; Murakami, Satoshi; Inoue, Tsuyoshi; Mori, Yusuke; Takano, Kazufumi

    2016-05-01

    We developed a new protein crystallization method that incorporates paper. A small piece of paper, such as facial tissue or KimWipes, was added to a drop of protein solution in the traditional sitting drop vapor diffusion technique, and protein crystals grew by incorporating paper. By this method, we achieved the growth of protein crystals with reducing osmotic shock. Because the technique is very simple and the materials are easy to obtain, this method will come into wide use for protein crystallization. In the future, it could be applied to nanoliter-scale crystallization screening on a paper sheet such as in inkjet printing.

  15. The skinny on CCN2.

    PubMed

    Leask, Andrew

    2008-12-01

    The CCN family of matricellular proteins directly or indirectly affects development and differentiation. A recent report written by Tan and colleagues (Am J Physiol Cell Physiol 295: C740-C751 2008) shows that CCN2 inhibits adipocyte differentiation. This commentary summarizes these observations.

  16. Forces stabilizing proteins.

    PubMed

    Nick Pace, C; Scholtz, J Martin; Grimsley, Gerald R

    2014-06-27

    The goal of this article is to summarize what has been learned about the major forces stabilizing proteins since the late 1980s when site-directed mutagenesis became possible. The following conclusions are derived from experimental studies of hydrophobic and hydrogen bonding variants. (1) Based on studies of 138 hydrophobic interaction variants in 11 proteins, burying a -CH2- group on folding contributes 1.1±0.5 kcal/mol to protein stability. (2) The burial of non-polar side chains contributes to protein stability in two ways: first, a term that depends on the removal of the side chains from water and, more importantly, the enhanced London dispersion forces that result from the tight packing in the protein interior. (3) Based on studies of 151 hydrogen bonding variants in 15 proteins, forming a hydrogen bond on folding contributes 1.1±0.8 kcal/mol to protein stability. (4) The contribution of hydrogen bonds to protein stability is strongly context dependent. (5) Hydrogen bonds by side chains and peptide groups make similar contributions to protein stability. (6) Polar group burial can make a favorable contribution to protein stability even if the polar group is not hydrogen bonded. (7) Hydrophobic interactions and hydrogen bonds both make large contributions to protein stability.

  17. Clinical protein mass spectrometry.

    PubMed

    Scherl, Alexander

    2015-06-15

    Quantitative protein analysis is routinely performed in clinical chemistry laboratories for diagnosis, therapeutic monitoring, and prognosis. Today, protein assays are mostly performed either with non-specific detection methods or immunoassays. Mass spectrometry (MS) is a very specific analytical method potentially very well suited for clinical laboratories. Its unique advantage relies in the high specificity of the detection. Any protein sequence variant, the presence of a post-translational modification or degradation will differ in mass and structure, and these differences will appear in the mass spectrum of the protein. On the other hand, protein MS is a relatively young technique, demanding specialized personnel and expensive instrumentation. Many scientists and opinion leaders predict MS to replace immunoassays for routine protein analysis, but there are only few protein MS applications routinely used in clinical chemistry laboratories today. The present review consists of a didactical introduction summarizing the pros and cons of MS assays compared to immunoassays, the different instrumentations, and various MS protein assays that have been proposed and/or are used in clinical laboratories. An important distinction is made between full length protein analysis (top-down method) and peptide analysis after enzymatic digestion of the proteins (bottom-up method) and its implication for the protein assay. The document ends with an outlook on what type of analyses could be used in the future, and for what type of applications MS has a clear advantage compared to immunoassays.

  18. Forces Stabilizing Proteins

    PubMed Central

    Pace, C. Nick; Scholtz, J. Martin; Grimsley, Gerald R.

    2014-01-01

    The goal of this article is to summarize what has been learned about the major forces stabilizing proteins since the late 1980s when site-directed mutagenesis became possible. The following conclusions are derived from experimental studies of hydrophobic and hydrogen bonding variants. 1. Based on studies of 138 hydrophobic interaction variants in 11 proteins, burying a –CH2– group on folding contributes 1.1 ± 0.5 kcal/mol to protein stability. 2. The burial of nonpolar side chains contributes to protein stability in two ways: first, a term that depends on the removal of the side chains from water and, more importantly, the enhanced London dispersion forces that result from the tight packing in the protein interior. 3. Based on studies of 151 hydrogen bonding variants in 15 proteins, forming a hydrogen bond on folding contributes 1.1 ± 0.8 kcal/mol to protein stability. 4. The contribution of hydrogen bonds to protein stability is strongly context dependent. 5. Hydrogen bonds by side chains and peptide groups make similar contributions to protein stability. 6. Polar group burial can make a favorable contribution to protein stability even if the polar group is not hydrogen bonded. 7. Hydrophobic interactions and hydrogen bonds both make large contributions to protein stability. PMID:24846139

  19. Protein Complexes in Bacteria

    PubMed Central

    Caufield, J. Harry; Abreu, Marco; Wimble, Christopher; Uetz, Peter

    2015-01-01

    Large-scale analyses of protein complexes have recently become available for Escherichia coli and Mycoplasma pneumoniae, yielding 443 and 116 heteromultimeric soluble protein complexes, respectively. We have coupled the results of these mass spectrometry-characterized protein complexes with the 285 “gold standard” protein complexes identified by EcoCyc. A comparison with databases of gene orthology, conservation, and essentiality identified proteins conserved or lost in complexes of other species. For instance, of 285 “gold standard” protein complexes in E. coli, less than 10% are fully conserved among a set of 7 distantly-related bacterial “model” species. Complex conservation follows one of three models: well-conserved complexes, complexes with a conserved core, and complexes with partial conservation but no conserved core. Expanding the comparison to 894 distinct bacterial genomes illustrates fractional conservation and the limits of co-conservation among components of protein complexes: just 14 out of 285 model protein complexes are perfectly conserved across 95% of the genomes used, yet we predict more than 180 may be partially conserved across at least half of the genomes. No clear relationship between gene essentiality and protein complex conservation is observed, as even poorly conserved complexes contain a significant number of essential proteins. Finally, we identify 183 complexes containing well-conserved components and uncharacterized proteins which will be interesting targets for future experimental studies. PMID:25723151

  20. Protein solubility modeling

    NASA Technical Reports Server (NTRS)

    Agena, S. M.; Pusey, M. L.; Bogle, I. D.

    1999-01-01

    A thermodynamic framework (UNIQUAC model with temperature dependent parameters) is applied to model the salt-induced protein crystallization equilibrium, i.e., protein solubility. The framework introduces a term for the solubility product describing protein transfer between the liquid and solid phase and a term for the solution behavior describing deviation from ideal solution. Protein solubility is modeled as a function of salt concentration and temperature for a four-component system consisting of a protein, pseudo solvent (water and buffer), cation, and anion (salt). Two different systems, lysozyme with sodium chloride and concanavalin A with ammonium sulfate, are investigated. Comparison of the modeled and experimental protein solubility data results in an average root mean square deviation of 5.8%, demonstrating that the model closely follows the experimental behavior. Model calculations and model parameters are reviewed to examine the model and protein crystallization process. Copyright 1999 John Wiley & Sons, Inc.

  1. Protein and vegetarian diets.

    PubMed

    Marsh, Kate A; Munn, Elizabeth A; Baines, Surinder K

    2013-08-19

    A vegetarian diet can easily meet human dietary protein requirements as long as energy needs are met and a variety of foods are eaten. Vegetarians should obtain protein from a variety of plant sources, including legumes, soy products, grains, nuts and seeds. Eggs and dairy products also provide protein for those following a lacto-ovo-vegetarian diet. There is no need to consciously combine different plant proteins at each meal as long as a variety of foods are eaten from day to day, because the human body maintains a pool of amino acids which can be used to complement dietary protein. The consumption of plant proteins rather than animal proteins by vegetarians may contribute to their reduced risk of chronic diseases such as diabetes and heart disease.

  2. Racemic protein crystallography.

    PubMed

    Yeates, Todd O; Kent, Stephen B H

    2012-01-01

    Although natural proteins are chiral and are all of one "handedness," their mirror image forms can be prepared by chemical synthesis. This opens up new opportunities for protein crystallography. A racemic mixture of the enantiomeric forms of a protein molecule can crystallize in ways that natural proteins cannot. Recent experimental data support a theoretical prediction that this should make racemic protein mixtures highly amenable to crystallization. Crystals obtained from racemic mixtures also offer advantages in structure determination strategies. The relevance of these potential advantages is heightened by advances in synthetic methods, which are extending the size limit for proteins that can be prepared by chemical synthesis. Recent ideas and results in the area of racemic protein crystallography are reviewed.

  3. Pigment-protein complexes

    SciTech Connect

    Siegelman, H W

    1980-01-01

    The photosynthetically-active pigment protein complexes of procaryotes and eucaryotes include chlorophyll proteins, carotenochlorophyll proteins, and biliproteins. They are either integral components or attached to photosynthetic membranes. Detergents are frequently required to solubilize the pigment-protein complexes. The membrane localization and detergent solubilization strongly suggest that the pigment-protein complexes are bound to the membranes by hydrophobic interactions. Hydrophobic interactions of proteins are characterized by an increase in entropy. Their bonding energy is directly related to temperature and ionic strength. Hydrophobic-interaction chromatography, a relatively new separation procedure, can furnish an important method for the purification of pigment-protein complexes. Phycobilisome purification and properties provide an example of the need to maintain hydrophobic interactions to preserve structure and function.

  4. Protein kinesis: The dynamics of protein trafficking and stability

    SciTech Connect

    1995-12-31

    The purpose of this conference is to provide a multidisciplinary forum for exchange of state-of-the-art information on protein kinesis. This volume contains abstracts of papers in the following areas: protein folding and modification in the endoplasmic reticulum; protein trafficking; protein translocation and folding; protein degradation; polarity; nuclear trafficking; membrane dynamics; and protein import into organelles.

  5. Toxic proteins in plants.

    PubMed

    Dang, Liuyi; Van Damme, Els J M

    2015-09-01

    Plants have evolved to synthesize a variety of noxious compounds to cope with unfavorable circumstances, among which a large group of toxic proteins that play a critical role in plant defense against predators and microbes. Up to now, a wide range of harmful proteins have been discovered in different plants, including lectins, ribosome-inactivating proteins, protease inhibitors, ureases, arcelins, antimicrobial peptides and pore-forming toxins. To fulfill their role in plant defense, these proteins exhibit various degrees of toxicity towards animals, insects, bacteria or fungi. Numerous studies have been carried out to investigate the toxic effects and mode of action of these plant proteins in order to explore their possible applications. Indeed, because of their biological activities, toxic plant proteins are also considered as potentially useful tools in crop protection and in biomedical applications, such as cancer treatment. Genes encoding toxic plant proteins have been introduced into crop genomes using genetic engineering technology in order to increase the plant's resistance against pathogens and diseases. Despite the availability of ample information on toxic plant proteins, very few publications have attempted to summarize the research progress made during the last decades. This review focuses on the diversity of toxic plant proteins in view of their toxicity as well as their mode of action. Furthermore, an outlook towards the biological role(s) of these proteins and their potential applications is discussed.

  6. Modeling Protein Expression and Protein Signaling Pathways

    PubMed Central

    Telesca, Donatello; Müller, Peter; Kornblau, Steven M.; Suchard, Marc A.; Ji, Yuan

    2015-01-01

    High-throughput functional proteomic technologies provide a way to quantify the expression of proteins of interest. Statistical inference centers on identifying the activation state of proteins and their patterns of molecular interaction formalized as dependence structure. Inference on dependence structure is particularly important when proteins are selected because they are part of a common molecular pathway. In that case, inference on dependence structure reveals properties of the underlying pathway. We propose a probability model that represents molecular interactions at the level of hidden binary latent variables that can be interpreted as indicators for active versus inactive states of the proteins. The proposed approach exploits available expert knowledge about the target pathway to define an informative prior on the hidden conditional dependence structure. An important feature of this prior is that it provides an instrument to explicitly anchor the model space to a set of interactions of interest, favoring a local search approach to model determination. We apply our model to reverse-phase protein array data from a study on acute myeloid leukemia. Our inference identifies relevant subpathways in relation to the unfolding of the biological process under study. PMID:26246646

  7. Energy design for protein-protein interactions

    PubMed Central

    Ravikant, D. V. S.; Elber, Ron

    2011-01-01

    Proteins bind to other proteins efficiently and specifically to carry on many cell functions such as signaling, activation, transport, enzymatic reactions, and more. To determine the geometry and strength of binding of a protein pair, an energy function is required. An algorithm to design an optimal energy function, based on empirical data of protein complexes, is proposed and applied. Emphasis is made on negative design in which incorrect geometries are presented to the algorithm that learns to avoid them. For the docking problem the search for plausible geometries can be performed exhaustively. The possible geometries of the complex are generated on a grid with the help of a fast Fourier transform algorithm. A novel formulation of negative design makes it possible to investigate iteratively hundreds of millions of negative examples while monotonically improving the quality of the potential. Experimental structures for 640 protein complexes are used to generate positive and negative examples for learning parameters. The algorithm designed in this work finds the correct binding structure as the lowest energy minimum in 318 cases of the 640 examples. Further benchmarks on independent sets confirm the significant capacity of the scoring function to recognize correct modes of interactions. PMID:21842951

  8. Cytoplasmic RNA modulators of an inside-out signal-transduction cascade

    PubMed Central

    Blind, Michael; Kolanus, Waldemar; Famulok, Michael

    1999-01-01

    A vaccinia virus-based RNA expression system enabled high-level cytoplasmic expression of RNA aptamers directed against the intracellular domain of the β2 integrin LFA-1, a transmembrane protein that mediates cell adhesion to intercellular adhesion molecule-1 (ICAM-1). In two different cell types, cytoplasmic expression of integrin-binding aptamers reduced inducible cell adhesion to ICAM-1. The aptamers specifically target, and thereby define, a functional cytoplasmic subdomain important for the regulation of cell adhesion in leukocytes. Our approach of aptamer-controlled blocking of signaling pathways in vivo could potentially be applied wherever targeted modulation of a signal-transduction cascade is desired. PMID:10097084

  9. Principles of Flexible Protein-Protein Docking

    PubMed Central

    Andrusier, Nelly; Mashiach, Efrat; Nussinov, Ruth; Wolfson, Haim J.

    2008-01-01

    Treating flexibility in molecular docking is a major challenge in cell biology research. Here we describe the background and the principles of existing flexible protein-protein docking methods, focusing on the algorithms and their rational. We describe how protein flexibility is treated in different stages of the docking process: in the preprocessing stage, rigid and flexible parts are identified and their possible conformations are modeled. This preprocessing provides information for the subsequent docking and refinement stages. In the docking stage, an ensemble of pre-generated conformations or the identified rigid domains may be docked separately. In the refinement stage, small-scale movements of the backbone and side-chains are modeled and the binding orientation is improved by rigid-body adjustments. For clarity of presentation, we divide the different methods into categories. This should allow the reader to focus on the most suitable method for a particular docking problem. PMID:18655061

  10. Antimicrobial proteins: From old proteins, new tricks.

    PubMed

    Smith, Valerie J; Dyrynda, Elisabeth A

    2015-12-01

    This review describes the main types of antimicrobial peptides (AMPs) synthesised by crustaceans, primarily those identified in shrimp, crayfish, crab and lobster. It includes an overview of their range of microbicidal activities and the current landscape of our understanding of their gene expression patterns in different body tissues. It further summarises how their expression might change following various types of immune challenges. The review further considers proteins or protein fragments from crustaceans that have antimicrobial properties but are more usually associated with other biological functions, or are derived from such proteins. It discusses how these unconventional AMPs might be generated at, or delivered to, sites of infection and how they might contribute to crustacean host defence in vivo. It also highlights recent work that is starting to reveal the extent of multi-functionality displayed by some decapod AMPs, particularly their participation in other aspects of host protection. Examples of such activities include proteinase inhibition, phagocytosis, antiviral activity and haematopoiesis. PMID:26320628

  11. Electrophoretic separation of proteins.

    PubMed

    Chakavarti, Bulbul; Chakavarti, Deb

    2008-01-01

    Electrophoresis is used to separate complex mixtures of proteins (e.g., from cells, subcellular fractions, column fractions, or immunoprecipitates), to investigate subunit compositions, and to verify homogeneity of protein samples. It can also serve to purify proteins for use in further applications. In polyacrylamide gel electrophoresis, proteins migrate in response to an electrical field through pores in a polyacrylamide gel matrix; pore size decreases with increasing acrylamide concentration. The combination of pore size and protein charge, size, and shape determines the migration rate of the protein. In this unit, the standard Laemmli method is described for discontinuous gel electrophoresis under denaturing conditions, i.e., in the presence of sodium dodecyl sulfate (SDS). PMID:19066548

  12. Functional Protein Microarray Technology

    PubMed Central

    Hu, Shaohui; Xie, Zhi; Qian, Jiang; Blackshaw, Seth; Zhu, Heng

    2010-01-01

    Functional protein microarrays are emerging as a promising new tool for large-scale and high-throughput studies. In this article, we will review their applications in basic proteomics research, where various types of assays have been developed to probe binding activities to other biomolecules, such as proteins, DNA, RNA, small molecules, and glycans. We will also report recent progress of using functional protein microarrays in profiling protein posttranslational modifications, including phosphorylation, ubiquitylation, acetylation, and nitrosylation. Finally, we will discuss potential of functional protein microarrays in biomarker identification and clinical diagnostics. We strongly believe that functional protein microarrays will soon become an indispensible and invaluable tool in proteomics research and systems biology. PMID:20872749

  13. Biofilm Matrix Proteins

    PubMed Central

    Fong, Jiunn N. C.; Yildiz, Fitnat H.

    2015-01-01

    Proteinaceous components of the biofilm matrix include secreted extracellular proteins, cell surface adhesins and protein subunits of cell appendages such as flagella and pili. Biofilm matrix proteins play diverse roles in biofilm formation and dissolution. They are involved in attaching cells to surfaces, stabilizing the biofilm matrix via interactions with exopolysaccharide and nucleic acid components, developing three-dimensional biofilm architectures, and dissolving biofilm matrix via enzymatic degradation of polysaccharides, proteins, and nucleic acids. In this chapter, we will review functions of matrix proteins in a selected set of microorganisms, studies of the matrix proteomes of Vibrio cholerae and Pseudomonas aeruginosa, and roles of outer membrane vesicles and of nucleoid-binding proteins in biofilm formation. PMID:26104709

  14. Protein Crystal Quality Studies

    NASA Technical Reports Server (NTRS)

    1998-01-01

    Eddie Snell, Post-Doctoral Fellow the National Research Council (NRC) uses a reciprocal space mapping diffractometer for macromolecular crystal quality studies. The diffractometer is used in mapping the structure of macromolecules such as proteins to determine their structure and thus understand how they function with other proteins in the body. This is one of several analytical tools used on proteins crystallized on Earth and in space experiments. Photo credit: NASA/Marshall Space Flight Center (MSFC)

  15. Computer Models of Proteins

    NASA Technical Reports Server (NTRS)

    2000-01-01

    Dr. Marc Pusey (seated) and Dr. Craig Kundrot use computers to analyze x-ray maps and generate three-dimensional models of protein structures. With this information, scientists at Marshall Space Flight Center can learn how proteins are made and how they work. The computer screen depicts a proten structure as a ball-and-stick model. Other models depict the actual volume occupied by the atoms, or the ribbon-like structures that are crucial to a protein's function.

  16. Protein oxidation and peroxidation.

    PubMed

    Davies, Michael J

    2016-04-01

    Proteins are major targets for radicals and two-electron oxidants in biological systems due to their abundance and high rate constants for reaction. With highly reactive radicals damage occurs at multiple side-chain and backbone sites. Less reactive species show greater selectivity with regard to the residues targeted and their spatial location. Modification can result in increased side-chain hydrophilicity, side-chain and backbone fragmentation, aggregation via covalent cross-linking or hydrophobic interactions, protein unfolding and altered conformation, altered interactions with biological partners and modified turnover. In the presence of O2, high yields of peroxyl radicals and peroxides (protein peroxidation) are formed; the latter account for up to 70% of the initial oxidant flux. Protein peroxides can oxidize both proteins and other targets. One-electron reduction results in additional radicals and chain reactions with alcohols and carbonyls as major products; the latter are commonly used markers of protein damage. Direct oxidation of cysteine (and less commonly) methionine residues is a major reaction; this is typically faster than with H2O2, and results in altered protein activity and function. Unlike H2O2, which is rapidly removed by protective enzymes, protein peroxides are only slowly removed, and catabolism is a major fate. Although turnover of modified proteins by proteasomal and lysosomal enzymes, and other proteases (e.g. mitochondrial Lon), can be efficient, protein hydroperoxides inhibit these pathways and this may contribute to the accumulation of modified proteins in cells. Available evidence supports an association between protein oxidation and multiple human pathologies, but whether this link is causal remains to be established.

  17. Protein oxidation and peroxidation

    PubMed Central

    Davies, Michael J.

    2016-01-01

    Proteins are major targets for radicals and two-electron oxidants in biological systems due to their abundance and high rate constants for reaction. With highly reactive radicals damage occurs at multiple side-chain and backbone sites. Less reactive species show greater selectivity with regard to the residues targeted and their spatial location. Modification can result in increased side-chain hydrophilicity, side-chain and backbone fragmentation, aggregation via covalent cross-linking or hydrophobic interactions, protein unfolding and altered conformation, altered interactions with biological partners and modified turnover. In the presence of O2, high yields of peroxyl radicals and peroxides (protein peroxidation) are formed; the latter account for up to 70% of the initial oxidant flux. Protein peroxides can oxidize both proteins and other targets. One-electron reduction results in additional radicals and chain reactions with alcohols and carbonyls as major products; the latter are commonly used markers of protein damage. Direct oxidation of cysteine (and less commonly) methionine residues is a major reaction; this is typically faster than with H2O2, and results in altered protein activity and function. Unlike H2O2, which is rapidly removed by protective enzymes, protein peroxides are only slowly removed, and catabolism is a major fate. Although turnover of modified proteins by proteasomal and lysosomal enzymes, and other proteases (e.g. mitochondrial Lon), can be efficient, protein hydroperoxides inhibit these pathways and this may contribute to the accumulation of modified proteins in cells. Available evidence supports an association between protein oxidation and multiple human pathologies, but whether this link is causal remains to be established. PMID:27026395

  18. Pressure cryocooling protein crystals

    DOEpatents

    Kim, Chae Un; Gruner, Sol M.

    2011-10-04

    Preparation of cryocooled protein crystal is provided by use of helium pressurizing and cryocooling to obtain cryocooled protein crystal allowing collection of high resolution data and by heavier noble gas (krypton or xenon) binding followed by helium pressurizing and cryocooling to obtain cryocooled protein crystal for collection of high resolution data and SAD phasing simultaneously. The helium pressurizing is carried out on crystal coated to prevent dehydration or on crystal grown in aqueous solution in a capillary.

  19. Protein-Losing Gastroenteropathy

    PubMed Central

    Pops, Martin A.

    1966-01-01

    In the past 10 years with the development of improved methods, particularly radioisotope techniques, it has been demonstrated that a number of patients with gastrointestinal disease and depletion of plasma proteins become hypoproteinemic because of actual leakage of albumin and other plasma proteins into the lumen of the gastrointestinal tract. The site of protein leakage is variable depending on the underlying pathological state but the loss of protein-containing lymph through the gastrointestinal lymphatic channels seems to be the major mechanism for hypoproteinemia. It has become apparent that there exists a normal mechanism for secretion of plasma proteins into the gastrointestinal tract as part of the overall metabolism of the plasma proteins. When the process is exaggerated so that resynthesis of plasma protein cannot keep pace with its degradation, sometimes severe hypoproteinemia is the result. Such a pathological process has now been described in approximately 40 disease states. A review of all the techniques which can demonstrate gastroenteric protein loss reveals that there are no widely available quantitative tests but that accurate quantitation is not necessary for the diagnosis of protein losing gastroenteropathy. PMID:18730025

  20. Human Mitochondrial Protein Database

    National Institute of Standards and Technology Data Gateway

    SRD 131 Human Mitochondrial Protein Database (Web, free access)   The Human Mitochondrial Protein Database (HMPDb) provides comprehensive data on mitochondrial and human nuclear encoded proteins involved in mitochondrial biogenesis and function. This database consolidates information from SwissProt, LocusLink, Protein Data Bank (PDB), GenBank, Genome Database (GDB), Online Mendelian Inheritance in Man (OMIM), Human Mitochondrial Genome Database (mtDB), MITOMAP, Neuromuscular Disease Center and Human 2-D PAGE Databases. This database is intended as a tool not only to aid in studying the mitochondrion but in studying the associated diseases.

  1. Acanthamoeba castellanii STAT protein.

    PubMed

    Kicinska, Anna; Leluk, Jacek; Jarmuszkiewicz, Wieslawa

    2014-01-01

    STAT (signal transducers and activators of transcription) proteins are one of the important mediators of phosphotyrosine-regulated signaling in metazoan cells. We described the presence of STAT protein in a unicellular, free-living amoebae with a simple life cycle, Acanthamoeba castellanii. A. castellanii is the only, studied to date, Amoebozoan that does not belong to Mycetozoa but possesses STATs. A sequence of the A. castellanii STAT protein includes domains similar to those of the Dictyostelium STAT proteins: a coiled coil (characteristic for Dictyostelium STAT coiled coil), a STAT DNA-binding domain and a Src-homology domain. The search for protein sequences homologous to A. castellanii STAT revealed 17 additional sequences from lower eukaryotes. Interestingly, all of these sequences come from Amoebozoa organisms that belong to either Mycetozoa (slime molds) or Centramoebida. We showed that there are four separated clades within the slime mold STAT proteins. The A. castellanii STAT protein branches next to a group of STATc proteins from Mycetozoa. We also demonstrate that Amoebozoa form a distinct monophyletic lineage within the STAT protein world that is well separated from the other groups. PMID:25338074

  2. The Halophile protein database.

    PubMed

    Sharma, Naveen; Farooqi, Mohammad Samir; Chaturvedi, Krishna Kumar; Lal, Shashi Bhushan; Grover, Monendra; Rai, Anil; Pandey, Pankaj

    2014-01-01

    Halophilic archaea/bacteria adapt to different salt concentration, namely extreme, moderate and low. These type of adaptations may occur as a result of modification of protein structure and other changes in different cell organelles. Thus proteins may play an important role in the adaptation of halophilic archaea/bacteria to saline conditions. The Halophile protein database (HProtDB) is a systematic attempt to document the biochemical and biophysical properties of proteins from halophilic archaea/bacteria which may be involved in adaptation of these organisms to saline conditions. In this database, various physicochemical properties such as molecular weight, theoretical pI, amino acid composition, atomic composition, estimated half-life, instability index, aliphatic index and grand average of hydropathicity (Gravy) have been listed. These physicochemical properties play an important role in identifying the protein structure, bonding pattern and function of the specific proteins. This database is comprehensive, manually curated, non-redundant catalogue of proteins. The database currently contains 59 897 proteins properties extracted from 21 different strains of halophilic archaea/bacteria. The database can be accessed through link. Database URL: http://webapp.cabgrid.res.in/protein/

  3. Moonlighting proteins in cancer.

    PubMed

    Min, Kyung-Won; Lee, Seong-Ho; Baek, Seung Joon

    2016-01-01

    Since the 1980s, growing evidence suggested that the cellular localization of proteins determined their activity and biological functions. In a classical view, a protein is characterized by the single cellular compartment where it primarily resides and functions. It is now believed that when proteins appear in different subcellular locations, the cells surpass the expected activity of proteins given the same genomic information to fulfill complex biological behavior. Many proteins are recognized for having the potential to exist in multiple locations in cells. Dysregulation of translocation may cause cancer or contribute to poorer cancer prognosis. Thus, quantitative and comprehensive assessment of dynamic proteins and associated protein movements could be a promising indicator in determining cancer prognosis and efficiency of cancer treatment and therapy. This review will summarize these so-called moonlighting proteins, in terms of a coupled intracellular cancer signaling pathway. Determination of the detailed biological intracellular and extracellular transit and regulatory activity of moonlighting proteins permits a better understanding of cancer and identification of potential means of molecular intervention.

  4. Biomolecular membrane protein crystallization

    NASA Astrophysics Data System (ADS)

    Reddy Bolla, Jani; Su, Chih-Chia; Yu, Edward W.

    2012-07-01

    Integral membrane proteins comprise approximately 30% of the sequenced genomes, and there is an immediate need for their high-resolution structural information. Currently, the most reliable approach to obtain these structures is X-ray crystallography. However, obtaining crystals of membrane proteins that diffract to high resolution appears to be quite challenging, and remains a major obstacle in structural determination. This brief review summarizes a variety of methodologies for use in crystallizing these membrane proteins. Hopefully, by introducing the available methods, techniques, and providing a general understanding of membrane proteins, a rational decision can be made about now to crystallize these complex materials.

  5. Glycolipid transfer proteins

    PubMed Central

    Brown, Rhoderick E.; Mattjus, Peter

    2007-01-01

    Glycolipid transfer proteins (GLTPs) are small (24 kD), soluble, ubiquitous proteins characterized by their ability to accelerate the intermembrane transfer of glycolipids in vitro. GLTP specificity encompasses both sphingoid- and glycerol-based glycolipids, but with a strict requirement that the initial sugar residue be beta-linked to the hydrophobic lipid backbone. The 3D protein structures of GLTP reveal liganded structures with unique lipid binding modes. The biochemical properties of GLTP action at the membrane surface have been studied rather comprehensively, but the biological role of GLTP remains enigmatic. What is clear is that GLTP differs distinctly from other known glycolipid-binding proteins, such as nonspecific lipid transfer proteins, lysosomal sphingolipid activator proteins, lectins, lung surfactant proteins as well as other lipid binding/transfer proteins. Based on the unique conformational architecture that targets GLTP to membranes and enables glycolipid binding, GLTP is now considered the prototypical and founding member of a new protein superfamily in eukaryotes. PMID:17320476

  6. Consensus protein design

    PubMed Central

    Porebski, Benjamin T.; Buckle, Ashley M.

    2016-01-01

    A popular and successful strategy in semi-rational design of protein stability is the use of evolutionary information encapsulated in homologous protein sequences. Consensus design is based on the hypothesis that at a given position, the respective consensus amino acid contributes more than average to the stability of the protein than non-conserved amino acids. Here, we review the consensus design approach, its theoretical underpinnings, successes, limitations and challenges, as well as providing a detailed guide to its application in protein engineering. PMID:27274091

  7. Chemical Synthesis of Proteins

    PubMed Central

    Nilsson, Bradley L.; Soellner, Matthew B.; Raines, Ronald T.

    2010-01-01

    Proteins have become accessible targets for chemical synthesis. The basic strategy is to use native chemical ligation, Staudinger ligation, or other orthogonal chemical reactions to couple synthetic peptides. The ligation reactions are compatible with a variety of solvents and proceed in solution or on a solid support. Chemical synthesis enables a level of control on protein composition that greatly exceeds that attainable with ribosome-mediated biosynthesis. Accordingly, the chemical synthesis of proteins is providing previously unattainable insight into the structure and function of proteins. PMID:15869385

  8. Self assembling proteins

    DOEpatents

    Yeates, Todd O.; Padilla, Jennifer; Colovos, Chris

    2004-06-29

    Novel fusion proteins capable of self-assembling into regular structures, as well as nucleic acids encoding the same, are provided. The subject fusion proteins comprise at least two oligomerization domains rigidly linked together, e.g. through an alpha helical linking group. Also provided are regular structures comprising a plurality of self-assembled fusion proteins of the subject invention, and methods for producing the same. The subject fusion proteins find use in the preparation of a variety of nanostructures, where such structures include: cages, shells, double-layer rings, two-dimensional layers, three-dimensional crystals, filaments, and tubes.

  9. Protein metabolism and requirements.

    PubMed

    Biolo, Gianni

    2013-01-01

    Skeletal muscle adaptation to critical illness includes insulin resistance, accelerated proteolysis, and increased release of glutamine and the other amino acids. Such amino acid efflux from skeletal muscle provides precursors for protein synthesis and energy fuel to the liver and to the rapidly dividing cells of the intestinal mucosa and the immune system. From these adaptation mechanisms, severe muscle wasting, glutamine depletion, and hyperglycemia, with increased patient morbidity and mortality, may ensue. Protein/amino acid nutrition, through either enteral or parenteral routes, plays a pivotal role in treatment of metabolic abnormalities in critical illness. In contrast to energy requirement, which can be accurately assessed by indirect calorimetry, methods to determine individual protein/amino acid needs are not currently available. In critical illness, a decreased ability of protein/amino acid intake to promote body protein synthesis is defined as anabolic resistance. This abnormality leads to increased protein/amino acid requirement and relative inefficiency of nutritional interventions. In addition to stress mediators, immobility and physical inactivity are key determinants of anabolic resistance. The development of mobility protocols in the intensive care unit should be encouraged to enhance the efficacy of nutrition. In critical illness, protein/amino acid requirement has been defined as the intake level associated with the lowest rate of catabolism. The optimal protein-sparing effects in patients receiving adequate energy are achieved when protein/amino acids are administered at rates between 1.3 and 1.5 g/kg/day. Extra glutamine supplementation is required in conditions of severe systemic inflammatory response. Protein requirement increases during hypocaloric feeding and in patients with acute renal failure on continuous renal replacement therapy. Evidence suggests that receiving adequate protein/amino acid intake may be more important than achieving

  10. Human Plasma Protein C

    PubMed Central

    Kisiel, Walter

    1979-01-01

    Protein C is a vitamin K-dependent protein, which exists in bovine plasma as a precursor of a serine protease. In this study, protein C was isolated to homogeneity from human plasma by barium citrate adsorption and elution, ammonium sulfate fractionation, DEAE-Sephadex chromatography, dextran sulfate agarose chromatography, and preparative polyacrylamide gel electrophoresis. Human protein C (Mr = 62,000) contains 23% carbohydrate and is composed of a light chain (Mr = 21,000) and a heavy chain (Mr = 41,000) held together by a disulfide bond(s). The light chain has an amino-terminal sequence of Ala-Asn-Ser-Phe-Leu- and the heavy chain has an aminoterminal sequence of Asp-Pro-Glu-Asp-Gln. The residues that are identical to bovine protein C are underlined. Incubation of human protein C with human α-thrombin at an enzyme to substrate weight ratio of 1:50 resulted in the formation of activated protein C, an enzyme with serine amidase activity. In the activation reaction, the apparent molecular weight of the heavy chain decreased from 41,000 to 40,000 as determined by gel electrophoresis in the presence of sodium dodecyl sulfate. No apparent change in the molecular weight of the light chain was observed in the activation process. The heavy chain of human activated protein C also contains the active-site serine residue as evidenced by its ability to react with radiolabeled diisopropyl fluorophosphate. Human activated protein C markedly prolongs the kaolin-cephalin clotting time of human plasma, but not that of bovine plasma. The amidolytic and anticoagulant activities of human activated protein C were completely obviated by prior incubation of the enzyme with diisopropyl fluorophosphate. These results indicate that human protein C, like its bovine counterpart, exists in plasma as a zymogen and is converted to a serine protease by limited proteolysis with attendant anticoagulant activity. Images PMID:468991

  11. Interolog interfaces in protein-protein docking.

    PubMed

    Alsop, James D; Mitchell, Julie C

    2015-11-01

    Proteins are essential elements of biological systems, and their function typically relies on their ability to successfully bind to specific partners. Recently, an emphasis of study into protein interactions has been on hot spots, or residues in the binding interface that make a significant contribution to the binding energetics. In this study, we investigate how conservation of hot spots can be used to guide docking prediction. We show that the use of evolutionary data combined with hot spot prediction highlights near-native structures across a range of benchmark examples. Our approach explores various strategies for using hot spots and evolutionary data to score protein complexes, using both absolute and chemical definitions of conservation along with refinements to these strategies that look at windowed conservation and filtering to ensure a minimum number of hot spots in each binding partner. Finally, structure-based models of orthologs were generated for comparison with sequence-based scoring. Using two data sets of 22 and 85 examples, a high rate of top 10 and top 1 predictions are observed, with up to 82% of examples returning a top 10 hit and 35% returning top 1 hit depending on the data set and strategy applied; upon inclusion of the native structure among the decoys, up to 55% of examples yielded a top 1 hit. The 20 common examples between data sets show that more carefully curated interolog data yields better predictions, particularly in achieving top 1 hits. Proteins 2015; 83:1940-1946. © 2015 The Authors. Proteins: Structure, Function, and Bioinformatics Published by Wiley Periodicals, Inc.

  12. The folate binding proteins.

    PubMed

    Corrocher, R; Olivieri, O; Pacor, M L

    1991-01-01

    Folates are essential molecules for cell life and, not surprisingly, their transport in biological fluids and their transfer to cells are finely regulated. Folate binding proteins play a major role in this regulation. This paper will review our knowledge on these proteins and examine the most recent advances in this field. PMID:1820987

  13. Poxviral Ankyrin Proteins

    PubMed Central

    Herbert, Michael H.; Squire, Christopher J.; Mercer, Andrew A

    2015-01-01

    Multiple repeats of the ankyrin motif (ANK) are ubiquitous throughout the kingdoms of life but are absent from most viruses. The main exception to this is the poxvirus family, and specifically the chordopoxviruses, with ANK repeat proteins present in all but three species from separate genera. The poxviral ANK repeat proteins belong to distinct orthologue groups spread over different species, and align well with the phylogeny of their genera. This distribution throughout the chordopoxviruses indicates these proteins were present in an ancestral vertebrate poxvirus, and have since undergone numerous duplication events. Most poxviral ANK repeat proteins contain an unusual topology of multiple ANK motifs starting at the N-terminus with a C-terminal poxviral homologue of the cellular F-box enabling interaction with the cellular SCF ubiquitin ligase complex. The subtle variations between ANK repeat proteins of individual poxviruses suggest an array of different substrates may be bound by these protein-protein interaction domains and, via the F-box, potentially directed to cellular ubiquitination pathways and possible degradation. Known interaction partners of several of these proteins indicate that the NF-κB coordinated anti-viral response is a key target, whilst some poxviral ANK repeat domains also have an F-box independent affect on viral host-range. PMID:25690795

  14. Protein Unfolding and Alzheimer's

    NASA Astrophysics Data System (ADS)

    Cheng, Kelvin

    2012-10-01

    Early interaction events of beta-amyloid (Aβ) proteins with neurons have been associated with the pathogenesis of Alzheimer's disease. Knowledge pertaining to the role of lipid molecules, particularly cholesterol, in modulating the single Aβ interactions with neurons at the atomic length and picosecond time resolutions, remains unclear. In our research, we have used atomistic molecular dynamics simulations to explore early molecular events including protein insertion kinetics, protein unfolding, and protein-induced membrane disruption of Aβ in lipid domains that mimic the nanoscopic raft and non-raft regions of the neural membrane. In this talk, I will summarize our current work on investigating the role of cholesterol in regulating the Aβ interaction events with membranes at the molecular level. I will also explain how our results will provide new insights into understanding the pathogenesis of Alzheimer's disease associated with the Aβ proteins.

  15. Structures of membrane proteins

    PubMed Central

    Vinothkumar, Kutti R.; Henderson, Richard

    2010-01-01

    In reviewing the structures of membrane proteins determined up to the end of 2009, we present in words and pictures the most informative examples from each family. We group the structures together according to their function and architecture to provide an overview of the major principles and variations on the most common themes. The first structures, determined 20 years ago, were those of naturally abundant proteins with limited conformational variability, and each membrane protein structure determined was a major landmark. With the advent of complete genome sequences and efficient expression systems, there has been an explosion in the rate of membrane protein structure determination, with many classes represented. New structures are published every month and more than 150 unique membrane protein structures have been determined. This review analyses the reasons for this success, discusses the challenges that still lie ahead, and presents a concise summary of the key achievements with illustrated examples selected from each class. PMID:20667175

  16. Protein disulfide engineering.

    PubMed

    Dombkowski, Alan A; Sultana, Kazi Zakia; Craig, Douglas B

    2014-01-21

    Improving the stability of proteins is an important goal in many biomedical and industrial applications. A logical approach is to emulate stabilizing molecular interactions found in nature. Disulfide bonds are covalent interactions that provide substantial stability to many proteins and conform to well-defined geometric conformations, thus making them appealing candidates in protein engineering efforts. Disulfide engineering is the directed design of novel disulfide bonds into target proteins. This important biotechnological tool has achieved considerable success in a wide range of applications, yet the rules that govern the stabilizing effects of disulfide bonds are not fully characterized. Contrary to expectations, many designed disulfide bonds have resulted in decreased stability of the modified protein. We review progress in disulfide engineering, with an emphasis on the issue of stability and computational methods that facilitate engineering efforts.

  17. Proteins, fluctuations and complexity

    SciTech Connect

    Frauenfelder, Hans; Chen, Guo; Fenimore, Paul W

    2008-01-01

    Glasses, supercooled liquids, and proteins share common properties, in particular the existence of two different types of fluctuations, {alpha} and {beta}. While the effect of the {alpha} fluctuations on proteins has been known for a few years, the effect of {beta} fluctuations has not been understood. By comparing neutron scattering data on the protein myoglobin with the {beta} fluctuations in the hydration shell measured by dielectric spectroscopy we show that the internal protein motions are slaved to these fluctuations. We also show that there is no 'dynamic transition' in proteins near 200 K. The rapid increase in the mean square displacement with temperature in many neutron scattering experiments is quantitatively predicted by the {beta} fluctuations in the hydration shell.

  18. Junin virus structural proteins.

    PubMed Central

    De Martínez Segovia, Z M; De Mitri, M I

    1977-01-01

    Polyacrylamide gel electrophoresis of purified Junin virus revealed six distinct structural polypeptides, two major and four minor ones. Four of these polypeptides appeared to be covalently linked with carbohydrate. The molecular weights of the six proteins, estimated by coelectrophoresis with marker proteins, ranged from 25,000 to 91,000. One of the two major components (number 3) was identified as a nucleoprotein and had a molecular weight of 64,000. It was the most prominent protein and was nonglycosylated. The other major protein (number 5), with a molecular weight of 38,000, was a glucoprotein and a component of the viral envelope. The location on the virion of three additional glycopeptides with molecular weights of 91,000, 72,000, and 52,000, together with a protein with a molecular weight of 25,000, was not well defined. PMID:189088

  19. Drugging Membrane Protein Interactions

    PubMed Central

    Yin, Hang; Flynn, Aaron D.

    2016-01-01

    The majority of therapeutics target membrane proteins, accessible on the surface of cells, to alter cellular signaling. Cells use membrane proteins to transduce signals into cells, transport ions and molecules, bind the cell to a surface or substrate, and catalyze reactions. Newly devised technologies allow us to drug conventionally “undruggable” regions of membrane proteins, enabling modulation of protein–protein, protein–lipid, and protein–nucleic acid interactions. In this review, we survey the state of the art in high-throughput screening and rational design in drug discovery, and we evaluate the advances in biological understanding and technological capacity that will drive pharmacotherapy forward against unorthodox membrane protein targets. PMID:26863923

  20. Proteins in unexpected locations.

    PubMed Central

    Smalheiser, N R

    1996-01-01

    Members of all classes of proteins--cytoskeletal components, secreted growth factors, glycolytic enzymes, kinases, transcription factors, chaperones, transmembrane proteins, and extracellular matrix proteins--have been identified in cellular compartments other than their conventional sites of action. Some of these proteins are expressed as distinct compartment-specific isoforms, have novel mechanisms for intercompartmental translocation, have distinct endogenous biological actions within each compartment, and are regulated in a compartment-specific manner as a function of physiologic state. The possibility that many, if not most, proteins have distinct roles in more than one cellular compartment has implications for the evolution of cell organization and may be important for understanding pathological conditions such as Alzheimer's disease and cancer. PMID:8862516

  1. Protein crystallization in microgravity.

    PubMed

    Aibara, S; Shibata, K; Morita, Y

    1997-12-01

    A space experiment involving protein crystallization was conducted in a microgravity environment using the space shuttle "Endeavour" of STS-47, on a 9-day mission from September 12th to 20th in 1992. The crystallization was carried out according to a batch method, and 5 proteins were selected as flight samples for crystallization. Two of these proteins: hen egg-white lysozyme and co-amino acid: pyruvate aminotransferase from Pseudomonas sp. F-126, were obtained as single crystals of good diffraction quality. Since 1992 we have carried out several space experiments for protein crystallization aboard space shuttles and the space station MIR. Our experimental results obtained mainly from hen egg-white lysozyme are described below, focusing on the effects of microgravity on protein crystal growth.

  2. Manipulating and Visualizing Proteins

    SciTech Connect

    Simon, Horst D.

    2003-12-05

    ProteinShop Gives Researchers a Hands-On Tool for Manipulating, Visualizing Protein Structures. The Human Genome Project and other biological research efforts are creating an avalanche of new data about the chemical makeup and genetic codes of living organisms. But in order to make sense of this raw data, researchers need software tools which let them explore and model data in a more intuitive fashion. With this in mind, researchers at Lawrence Berkeley National Laboratory and the University of California, Davis, have developed ProteinShop, a visualization and modeling program which allows researchers to manipulate protein structures with pinpoint control, guided in large part by their own biological and experimental instincts. Biologists have spent the last half century trying to unravel the ''protein folding problem,'' which refers to the way chains of amino acids physically fold themselves into three-dimensional proteins. This final shape, which resembles a crumpled ribbon or piece of origami, is what determines how the protein functions and translates genetic information. Understanding and modeling this geometrically complex formation is no easy matter. ProteinShop takes a given sequence of amino acids and uses visualization guides to help generate predictions about the secondary structures, identifying alpha helices and flat beta strands, and the coil regions that bind them. Once secondary structures are in place, researchers can twist and turn these pre-configurations until they come up with a number of possible tertiary structure conformations. In turn, these are fed into a computationally intensive optimization procedure that tries to find the final, three-dimensional protein structure. Most importantly, ProteinShop allows users to add human knowledge and intuition to the protein structure prediction process, thus bypassing bad configurations that would otherwise be fruitless for optimization. This saves compute cycles and accelerates the entire process, so

  3. Regulation of protein turnover by heat shock proteins.

    PubMed

    Bozaykut, Perinur; Ozer, Nesrin Kartal; Karademir, Betul

    2014-12-01

    Protein turnover reflects the balance between synthesis and degradation of proteins, and it is a crucial process for the maintenance of the cellular protein pool. The folding of proteins, refolding of misfolded proteins, and also degradation of misfolded and damaged proteins are involved in the protein quality control (PQC) system. Correct protein folding and degradation are controlled by many different factors, one of the most important of which is the heat shock protein family. Heat shock proteins (HSPs) are in the class of molecular chaperones, which may prevent the inappropriate interaction of proteins and induce correct folding. On the other hand, these proteins play significant roles in the degradation pathways, including endoplasmic reticulum-associated degradation (ERAD), the ubiquitin-proteasome system, and autophagy. This review focuses on the emerging role of HSPs in the regulation of protein turnover; the effects of HSPs on the degradation machineries ERAD, autophagy, and proteasome; as well as the role of posttranslational modifications in the PQC system.

  4. Protein crystal growth

    NASA Technical Reports Server (NTRS)

    Bugg, Charles E.

    1993-01-01

    Proteins account for 50% or more of the dry weight of most living systems and play a crucial role in virtually all biological processes. Since the specific functions of essentially all biological molecules are determined by their three-dimensional structures, it is obvious that a detailed understanding of the structural makeup of a protein is essential to any systematic research pertaining to it. At the present time, protein crystallography has no substitute, it is the only technique available for elucidating the atomic arrangements within complicated biological molecules. Most macromolecules are extremely difficult to crystallize, and many otherwise exciting and promising projects have terminated at the crystal growth stage. There is a pressing need to better understand protein crystal growth, and to develop new techniques that can be used to enhance the size and quality of protein crystals. There are several aspects of microgravity that might be exploited to enhance protein crystal growth. The major factor that might be expected to alter crystal growth processes in space is the elimination of density-driven convective flow. Another factor that can be readily controlled in the absence of gravity is the sedimentation of growing crystal in a gravitational field. Another potential advantage of microgravity for protein crystal growth is the option of doing containerless crystal growth. One can readily understand why the microgravity environment established by Earth-orbiting vehicles is perceived to offer unique opportunities for the protein crystallographer. The near term objectives of the Protein Crystal Growth in a Microgravity Environment (PCG/ME) project is to continue to improve the techniques, procedures, and hardware systems used to grow protein crystals in Earth orbit.

  5. Protein Binding Pocket Dynamics.

    PubMed

    Stank, Antonia; Kokh, Daria B; Fuller, Jonathan C; Wade, Rebecca C

    2016-05-17

    The dynamics of protein binding pockets are crucial for their interaction specificity. Structural flexibility allows proteins to adapt to their individual molecular binding partners and facilitates the binding process. This implies the necessity to consider protein internal motion in determining and predicting binding properties and in designing new binders. Although accounting for protein dynamics presents a challenge for computational approaches, it expands the structural and physicochemical space for compound design and thus offers the prospect of improved binding specificity and selectivity. A cavity on the surface or in the interior of a protein that possesses suitable properties for binding a ligand is usually referred to as a binding pocket. The set of amino acid residues around a binding pocket determines its physicochemical characteristics and, together with its shape and location in a protein, defines its functionality. Residues outside the binding site can also have a long-range effect on the properties of the binding pocket. Cavities with similar functionalities are often conserved across protein families. For example, enzyme active sites are usually concave surfaces that present amino acid residues in a suitable configuration for binding low molecular weight compounds. Macromolecular binding pockets, on the other hand, are located on the protein surface and are often shallower. The mobility of proteins allows the opening, closing, and adaptation of binding pockets to regulate binding processes and specific protein functionalities. For example, channels and tunnels can exist permanently or transiently to transport compounds to and from a binding site. The influence of protein flexibility on binding pockets can vary from small changes to an already existent pocket to the formation of a completely new pocket. Here, we review recent developments in computational methods to detect and define binding pockets and to study pocket dynamics. We introduce five

  6. Protein Binding Pocket Dynamics.

    PubMed

    Stank, Antonia; Kokh, Daria B; Fuller, Jonathan C; Wade, Rebecca C

    2016-05-17

    The dynamics of protein binding pockets are crucial for their interaction specificity. Structural flexibility allows proteins to adapt to their individual molecular binding partners and facilitates the binding process. This implies the necessity to consider protein internal motion in determining and predicting binding properties and in designing new binders. Although accounting for protein dynamics presents a challenge for computational approaches, it expands the structural and physicochemical space for compound design and thus offers the prospect of improved binding specificity and selectivity. A cavity on the surface or in the interior of a protein that possesses suitable properties for binding a ligand is usually referred to as a binding pocket. The set of amino acid residues around a binding pocket determines its physicochemical characteristics and, together with its shape and location in a protein, defines its functionality. Residues outside the binding site can also have a long-range effect on the properties of the binding pocket. Cavities with similar functionalities are often conserved across protein families. For example, enzyme active sites are usually concave surfaces that present amino acid residues in a suitable configuration for binding low molecular weight compounds. Macromolecular binding pockets, on the other hand, are located on the protein surface and are often shallower. The mobility of proteins allows the opening, closing, and adaptation of binding pockets to regulate binding processes and specific protein functionalities. For example, channels and tunnels can exist permanently or transiently to transport compounds to and from a binding site. The influence of protein flexibility on binding pockets can vary from small changes to an already existent pocket to the formation of a completely new pocket. Here, we review recent developments in computational methods to detect and define binding pockets and to study pocket dynamics. We introduce five

  7. Fullerene sorting proteins.

    PubMed

    Calvaresi, Matteo; Zerbetto, Francesco

    2011-07-01

    Proteins bind fullerenes. Hydrophobic pockets can accommodate a carbon cage either in full or in part. However, the identification of proteins able to discriminate between different cages is an open issue. Prediction of candidates able to perform this function is desirable and is achieved with an inverse docking procedure that accurately accounts for (i) van der Waals interactions between the cage and the protein surface, (ii) desolvation free energy, (iii) shape complementarity, and (iv) minimization of the number of steric clashes through conformational variations. A set of more than 1000 protein structures is divided into four categories that either select C(60) or C(70) (p-C(60) or p-C(70)) and either accommodate the cages in the same pocket (homosaccic proteins, from σακκoζ meaning pocket) or in different pockets (heterosaccic proteins). In agreement with the experiments, the KcsA Potassium Channel is predicted to have one of the best performances for both cages. Possible ways to exploit the results and efficiently separate the two cages with proteins are also discussed.

  8. NMCP/LINC proteins

    PubMed Central

    Ciska, Malgorzata; Moreno Díaz de la Espina, Susana

    2013-01-01

    Lamins are the main components of the metazoan lamina, and while the organization of the nuclear lamina of metazoans and plants is similar, there are apparently no genes encoding lamins or most lamin-binding proteins in plants. Thus, the plant lamina is not lamin-based and the proteins that form this structure are still to be characterized. Members of the plant NMCP/LINC/CRWN protein family share the typical tripartite structure of lamins, although the 2 exhibit no sequence similarity. However, given the many similarities between NMCP/LINC/CRWN proteins and lamins (structural organization, position of conserved regions, sub-nuclear distribution, solubility, and pattern of expression), these proteins are good candidates to carry out the functions of lamins in plants. Moreover, functional analysis of NMCP/LINC mutants has revealed their involvement in maintaining nuclear size and shape, another activity fulfilled by lamins. This review summarizes the current understanding of NMCP/LINC proteins and discusses future studies that will be required to demonstrate definitively that these proteins are plant analogs of lamins. PMID:24128696

  9. PSC: protein surface classification.

    PubMed

    Tseng, Yan Yuan; Li, Wen-Hsiung

    2012-07-01

    We recently proposed to classify proteins by their functional surfaces. Using the structural attributes of functional surfaces, we inferred the pairwise relationships of proteins and constructed an expandable database of protein surface classification (PSC). As the functional surface(s) of a protein is the local region where the protein performs its function, our classification may reflect the functional relationships among proteins. Currently, PSC contains a library of 1974 surface types that include 25,857 functional surfaces identified from 24,170 bound structures. The search tool in PSC empowers users to explore related surfaces that share similar local structures and core functions. Each functional surface is characterized by structural attributes, which are geometric, physicochemical or evolutionary features. The attributes have been normalized as descriptors and integrated to produce a profile for each functional surface in PSC. In addition, binding ligands are recorded for comparisons among homologs. PSC allows users to exploit related binding surfaces to reveal the changes in functionally important residues on homologs that have led to functional divergence during evolution. The substitutions at the key residues of a spatial pattern may determine the functional evolution of a protein. In PSC (http://pocket.uchicago.edu/psc/), a pool of changes in residues on similar functional surfaces is provided.

  10. Bacterial Ice Crystal Controlling Proteins

    PubMed Central

    Lorv, Janet S. H.; Rose, David R.; Glick, Bernard R.

    2014-01-01

    Across the world, many ice active bacteria utilize ice crystal controlling proteins for aid in freezing tolerance at subzero temperatures. Ice crystal controlling proteins include both antifreeze and ice nucleation proteins. Antifreeze proteins minimize freezing damage by inhibiting growth of large ice crystals, while ice nucleation proteins induce formation of embryonic ice crystals. Although both protein classes have differing functions, these proteins use the same ice binding mechanisms. Rather than direct binding, it is probable that these protein classes create an ice surface prior to ice crystal surface adsorption. Function is differentiated by molecular size of the protein. This paper reviews the similar and different aspects of bacterial antifreeze and ice nucleation proteins, the role of these proteins in freezing tolerance, prevalence of these proteins in psychrophiles, and current mechanisms of protein-ice interactions. PMID:24579057

  11. Bacterial ice crystal controlling proteins.

    PubMed

    Lorv, Janet S H; Rose, David R; Glick, Bernard R

    2014-01-01

    Across the world, many ice active bacteria utilize ice crystal controlling proteins for aid in freezing tolerance at subzero temperatures. Ice crystal controlling proteins include both antifreeze and ice nucleation proteins. Antifreeze proteins minimize freezing damage by inhibiting growth of large ice crystals, while ice nucleation proteins induce formation of embryonic ice crystals. Although both protein classes have differing functions, these proteins use the same ice binding mechanisms. Rather than direct binding, it is probable that these protein classes create an ice surface prior to ice crystal surface adsorption. Function is differentiated by molecular size of the protein. This paper reviews the similar and different aspects of bacterial antifreeze and ice nucleation proteins, the role of these proteins in freezing tolerance, prevalence of these proteins in psychrophiles, and current mechanisms of protein-ice interactions. PMID:24579057

  12. Protein microarrays: prospects and problems.

    PubMed

    Kodadek, T

    2001-02-01

    Protein microarrays are potentially powerful tools in biochemistry and molecular biology. Two types of protein microarrays are defined. One, termed a protein function array, will consist of thousands of native proteins immobilized in a defined pattern. Such arrays can be utilized for massively parallel testing of protein function, hence the name. The other type is termed a protein-detecting array. This will consist of large numbers of arrayed protein-binding agents. These arrays will allow for expression profiling to be done at the protein level. In this article, some of the major technological challenges to the development of protein arrays are discussed, along with potential solutions.

  13. Phospholipid transfer proteins revisited.

    PubMed Central

    Wirtz, K W

    1997-01-01

    Phosphatidylinositol transfer protein (PI-TP) and the non-specific lipid transfer protein (nsL-TP) (identical with sterol carrier protein 2) belong to the large and diverse family of intracellular lipid-binding proteins. Although these two proteins may express a comparable phospholipid transfer activity in vitro, recent studies in yeast and mammalian cells have indicated that they serve completely different functions. PI-TP (identical with yeast SEC14p) plays an important role in vesicle flow both in the budding reaction from the trans-Golgi network and in the fusion reaction with the plasma membrane. In yeast, vesicle budding is linked to PI-TP regulating Golgi phosphatidylcholine (PC) biosynthesis with the apparent purpose of maintaining an optimal PI/PC ratio of the Golgi complex. In mammalian cells, vesicle flow appears to be dependent on PI-TP stimulating phosphatidylinositol 4,5-bisphosphate (PIP2) synthesis. This latter process may also be linked to the ability of PI-TP to reconstitute the receptor-controlled PIP2-specific phospholipase C activity. The nsL-TP is a peroxisomal protein which, by its ability to bind fatty acyl-CoAs, is most likely involved in the beta-oxidation of fatty acids in this organelle. This protein constitutes the N-terminus of the 58 kDa protein which is one of the peroxisomal 3-oxo-acyl-CoA thiolases. Further studies on these and other known phospholipid transfer proteins are bound to reveal new insights in their important role as mediators between lipid metabolism and cell functions. PMID:9182690

  14. (PCG) Protein Crystal Growth Canavalin

    NASA Technical Reports Server (NTRS)

    1989-01-01

    (PCG) Protein Crystal Growth Canavalin. The major storage protein of leguminous plants and a major source of dietary protein for humans and domestic animals. It is studied in efforts to enhance nutritional value of proteins through protein engineerings. It is isolated from Jack Bean because of it's potential as a nutritional substance. Principal Investigator on STS-26 was Alex McPherson.

  15. Structure Prediction of Protein Complexes

    NASA Astrophysics Data System (ADS)

    Pierce, Brian; Weng, Zhiping

    Protein-protein interactions are critical for biological function. They directly and indirectly influence the biological systems of which they are a part. Antibodies bind with antigens to detect and stop viruses and other infectious agents. Cell signaling is performed in many cases through the interactions between proteins. Many diseases involve protein-protein interactions on some level, including cancer and prion diseases.

  16. Piezoelectric allostery of protein.

    PubMed

    Ohnuki, Jun; Sato, Takato; Takano, Mitsunori

    2016-07-01

    Allostery is indispensable for a protein to work, where a locally applied stimulus is transmitted to a distant part of the molecule. While the allostery due to chemical stimuli such as ligand binding has long been studied, the growing interest in mechanobiology prompts the study of the mechanically stimulated allostery, the physical mechanism of which has not been established. By molecular dynamics simulation of a motor protein myosin, we found that a locally applied mechanical stimulus induces electrostatic potential change at distant regions, just like the piezoelectricity. This novel allosteric mechanism, "piezoelectric allostery", should be of particularly high value for mechanosensor/transducer proteins. PMID:27575163

  17. Protein crystallography prescreen kit

    DOEpatents

    Segelke, Brent W.; Krupka, Heike I.; Rupp, Bernhard

    2007-10-02

    A kit for prescreening protein concentration for crystallization includes a multiplicity of vials, a multiplicity of pre-selected reagents, and a multiplicity of sample plates. The reagents and a corresponding multiplicity of samples of the protein in solutions of varying concentrations are placed on sample plates. The sample plates containing the reagents and samples are incubated. After incubation the sample plates are examined to determine which of the sample concentrations are too low and which the sample concentrations are too high. The sample concentrations that are optimal for protein crystallization are selected and used.

  18. Protein crystallography prescreen kit

    DOEpatents

    Segelke, Brent W.; Krupka, Heike I.; Rupp, Bernhard

    2005-07-12

    A kit for prescreening protein concentration for crystallization includes a multiplicity of vials, a multiplicity of pre-selected reagents, and a multiplicity of sample plates. The reagents and a corresponding multiplicity of samples of the protein in solutions of varying concentrations are placed on sample plates. The sample plates containing the reagents and samples are incubated. After incubation the sample plates are examined to determine which of the sample concentrations are too low and which the sample concentrations are too high. The sample concentrations that are optimal for protein crystallization are selected and used.

  19. Protein Crystal Malic Enzyme

    NASA Technical Reports Server (NTRS)

    1992-01-01

    Malic Enzyme is a target protein for drug design because it is a key protein in the life cycle of intestinal parasites. After 2 years of effort on Earth, investigators were unable to produce any crystals that were of high enough quality and for this reason the structure of this important protein could not be determined. Crystals obtained from one STS-50 were of superior quality allowing the structure to be determined. This is just one example why access to space is so vital for these studies. Principal Investigator is Larry DeLucas.

  20. Protein Crystal Quality Studies

    NASA Technical Reports Server (NTRS)

    1998-01-01

    Eddie Snell (standing), Post-Doctoral Fellow the National Research Council (NRC),and Marc Pusey of Marshall Space Flight Center (MSFC) use a reciprocal space mapping diffractometer for marcromolecular crystal quality studies. The diffractometer is used in mapping the structure of marcromolecules such as proteins to determine their structure and thus understand how they function with other proteins in the body. This is one of several analytical tools used on proteins crystalized on Earth and in space experiments. Photo credit: NASA/Marshall Space Flight Center (MSFC)

  1. Protein based Block Copolymers

    PubMed Central

    Rabotyagova, Olena S.; Cebe, Peggy; Kaplan, David L.

    2011-01-01

    Advances in genetic engineering have led to the synthesis of protein-based block copolymers with control of chemistry and molecular weight, resulting in unique physical and biological properties. The benefits from incorporating peptide blocks into copolymer designs arise from the fundamental properties of proteins to adopt ordered conformations and to undergo self-assembly, providing control over structure formation at various length scales when compared to conventional block copolymers. This review covers the synthesis, structure, assembly, properties, and applications of protein-based block copolymers. PMID:21235251

  2. Amino acids and proteins.

    PubMed

    van Goudoever, Johannes B; Vlaardingerbroek, Hester; van den Akker, Chris H; de Groof, Femke; van der Schoor, Sophie R D

    2014-01-01

    Amino acids and protein are key factors for growth. The neonatal period requires the highest intake in life to meet the demands. Those demands include amino acids for growth, but proteins and amino acids also function as signalling molecules and function as neurotransmitters. Often the nutritional requirements are not met, resulting in a postnatal growth restriction. However, current knowledge on adequate levels of both amino acid as well as protein intake can avoid under nutrition in the direct postnatal phase, avoid the need for subsequent catch-up growth and improve later outcome.

  3. Piezoelectric allostery of protein

    NASA Astrophysics Data System (ADS)

    Ohnuki, Jun; Sato, Takato; Takano, Mitsunori

    2016-07-01

    Allostery is indispensable for a protein to work, where a locally applied stimulus is transmitted to a distant part of the molecule. While the allostery due to chemical stimuli such as ligand binding has long been studied, the growing interest in mechanobiology prompts the study of the mechanically stimulated allostery, the physical mechanism of which has not been established. By molecular dynamics simulation of a motor protein myosin, we found that a locally applied mechanical stimulus induces electrostatic potential change at distant regions, just like the piezoelectricity. This novel allosteric mechanism, "piezoelectric allostery", should be of particularly high value for mechanosensor/transducer proteins.

  4. The protein-protein interaction map of Helicobacter pylori.

    PubMed

    Rain, J C; Selig, L; De Reuse, H; Battaglia, V; Reverdy, C; Simon, S; Lenzen, G; Petel, F; Wojcik, J; Schächter, V; Chemama, Y; Labigne, A; Legrain, P

    2001-01-11

    With the availability of complete DNA sequences for many prokaryotic and eukaryotic genomes, and soon for the human genome itself, it is important to develop reliable proteome-wide approaches for a better understanding of protein function. As elementary constituents of cellular protein complexes and pathways, protein-protein interactions are key determinants of protein function. Here we have built a large-scale protein-protein interaction map of the human gastric pathogen Helicobacter pylori. We have used a high-throughput strategy of the yeast two-hybrid assay to screen 261 H. pylori proteins against a highly complex library of genome-encoded polypeptides. Over 1,200 interactions were identified between H. pylori proteins, connecting 46.6% of the proteome. The determination of a reliability score for every single protein-protein interaction and the identification of the actual interacting domains permitted the assignment of unannotated proteins to biological pathways.

  5. Dietary Proteins and Angiogenesis

    PubMed Central

    Medina, Miguel Ángel; Quesada, Ana R.

    2014-01-01

    Both defective and persistent angiogenesis are linked to pathological situations in the adult. Compounds able to modulate angiogenesis have a potential value for the treatment of such pathologies. Several small molecules present in the diet have been shown to have modulatory effects on angiogenesis. This review presents the current state of knowledge on the potential modulatory roles of dietary proteins on angiogenesis. There is currently limited available information on the topic. Milk contains at least three proteins for which modulatory effects on angiogenesis have been previously demonstrated. On the other hand, there is some scarce information on the potential of dietary lectins, edible plant proteins and high protein diets to modulate angiogenesis. PMID:24445377

  6. Plant protein glycosylation

    PubMed Central

    Strasser, Richard

    2016-01-01

    Protein glycosylation is an essential co- and post-translational modification of secretory and membrane proteins in all eukaryotes. The initial steps of N-glycosylation and N-glycan processing are highly conserved between plants, mammals and yeast. In contrast, late N-glycan maturation steps in the Golgi differ significantly in plants giving rise to complex N-glycans with β1,2-linked xylose, core α1,3-linked fucose and Lewis A-type structures. While the essential role of N-glycan modifications on distinct mammalian glycoproteins is already well documented, we have only begun to decipher the biological function of this ubiquitous protein modification in different plant species. In this review, I focus on the biosynthesis and function of different protein N-linked glycans in plants. Special emphasis is given on glycan-mediated quality control processes in the ER and on the biological role of characteristic complex N-glycan structures. PMID:26911286

  7. Densonucleosis virus structural proteins.

    PubMed

    Kelly, D C; Moore, N F; Spilling, C R; Barwise, A H; Walker, I O

    1980-10-01

    The protein coats of two densonucleosis viruses (types 1 and 2) were examined by a variety of biophysical, biochemical, and serological techniques. The viruses were 24 nm in diameter, contained at least four polypeptides, were remarkably stable to extremes of pH and denaturing agents, and were serologically closely related. The two viruses could, however, be distinguished serologically and by differences in migration of their structural polypeptides. For each virus the "top component" (i.e., the protein coat minus DNA, found occurring naturally in infections) appeared to have a composition identical to that of the coat of the virus and was a more stable structure. Electrometric titration curves of the virus particles and top components demonstrated that the DNA phosphate in densonucleosis virus particles was neutralized by cations other than basic amino acid side chains of the protein coat. Circular dichroism studies showed that there was a conformational difference between the protein coats of top components and virus particles.

  8. Protein fabrication automation

    PubMed Central

    Cox, J. Colin; Lape, Janel; Sayed, Mahmood A.; Hellinga, Homme W.

    2007-01-01

    Facile “writing” of DNA fragments that encode entire gene sequences potentially has widespread applications in biological analysis and engineering. Rapid writing of open reading frames (ORFs) for expressed proteins could transform protein engineering and production for protein design, synthetic biology, and structural analysis. Here we present a process, protein fabrication automation (PFA), which facilitates the rapid de novo construction of any desired ORF from oligonucleotides with low effort, high speed, and little human interaction. PFA comprises software for sequence design, data management, and the generation of instruction sets for liquid-handling robotics, a liquid-handling robot, a robust PCR scheme for gene assembly from synthetic oligonucleotides, and a genetic selection system to enrich correctly assembled full-length synthetic ORFs. The process is robust and scalable. PMID:17242375

  9. Protein Colloidal Aggregation Project

    NASA Technical Reports Server (NTRS)

    Oliva-Buisson, Yvette J. (Compiler)

    2014-01-01

    To investigate the pathways and kinetics of protein aggregation to allow accurate predictive modeling of the process and evaluation of potential inhibitors to prevalent diseases including cataract formation, chronic traumatic encephalopathy, Alzheimer's Disease, Parkinson's Disease and others.

  10. C-reactive protein

    MedlinePlus

    ... body. It is one of a group of proteins called "acute phase reactants" that go up in response to inflammation. This article discusses the blood test done to measure the amount of CRP in your blood.

  11. Protein fabrication automation.

    PubMed

    Cox, J Colin; Lape, Janel; Sayed, Mahmood A; Hellinga, Homme W

    2007-03-01

    Facile "writing" of DNA fragments that encode entire gene sequences potentially has widespread applications in biological analysis and engineering. Rapid writing of open reading frames (ORFs) for expressed proteins could transform protein engineering and production for protein design, synthetic biology, and structural analysis. Here we present a process, protein fabrication automation (PFA), which facilitates the rapid de novo construction of any desired ORF from oligonucleotides with low effort, high speed, and little human interaction. PFA comprises software for sequence design, data management, and the generation of instruction sets for liquid-handling robotics, a liquid-handling robot, a robust PCR scheme for gene assembly from synthetic oligonucleotides, and a genetic selection system to enrich correctly assembled full-length synthetic ORFs. The process is robust and scalable.

  12. Protein conducting nanopores

    NASA Astrophysics Data System (ADS)

    Harsman, Anke; Krüger, Vivien; Bartsch, Philipp; Honigmann, Alf; Schmidt, Oliver; Rao, Sanjana; Meisinger, Christof; Wagner, Richard

    2010-11-01

    About 50% of the cellular proteins have to be transported into or across cellular membranes. This transport is an essential step in the protein biosynthesis. In eukaryotic cells secretory proteins are transported into the endoplasmic reticulum before they are transported in vesicles to the plasma membrane. Almost all proteins of the endosymbiotic organelles chloroplasts and mitochondria are synthesized on cytosolic ribosomes and posttranslationally imported. Genetic, biochemical and biophysical approaches led to rather detailed knowledge on the composition of the translocon-complexes which catalyze the membrane transport of the preproteins. Comprehensive concepts on the targeting and membrane transport of polypeptides emerged, however little detail on the molecular nature and mechanisms of the protein translocation channels comprising nanopores has been achieved. In this paper we will highlight recent developments of the diverse protein translocation systems and focus particularly on the common biophysical properties and functions of the protein conducting nanopores. We also provide a first analysis of the interaction between the genuine protein conducting nanopore Tom40SC as well as a mutant Tom40SC (\\mathrm {S}_{54} \\to E ) containing an additional negative charge at the channel vestibule and one of its native substrates, CoxIV, a mitochondrial targeting peptide. The polypeptide induced a voltage-dependent increase in the frequency of channel closure of Tom40SC corresponding to a voltage-dependent association rate, which was even more pronounced for the Tom40SC S54E mutant. The corresponding dwelltime reflecting association/transport of the peptide could be determined with \\bar {t}_{\\mathrm {off}} \\cong 1.1 ms for the wildtype, whereas the mutant Tom40SC S54E displayed a biphasic dwelltime distribution (\\bar {t}_{\\mathrm {off}}^1 \\cong 0.4 ms \\bar {t}_{\\mathrm {off}}^2 \\cong 4.6 ms).

  13. Recombinant Collagenlike Proteins

    NASA Technical Reports Server (NTRS)

    Fertala, Andzej

    2007-01-01

    A group of collagenlike recombinant proteins containing high densities of biologically active sites has been invented. The method used to express these proteins is similar to a method of expressing recombinant procollagens and collagens described in U. S. Patent 5,593,859, "Synthesis of human procollagens and collagens in recombinant DNA systems." Customized collagenous proteins are needed for biomedical applications. In particular, fibrillar collagens are attractive for production of matrices needed for tissue engineering and drug delivery. Prior to this invention, there was no way of producing customized collagenous proteins for these and other applications. Heretofore, collagenous proteins have been produced by use of such biological systems as yeasts, bacteria, and transgenic animals and plants. These products are normal collagens that can also be extracted from such sources as tendons, bones, and hides. These products cannot be made to consist only of biologically active, specific amino acid sequences that may be needed for specific applications. Prior to this invention, it had been established that fibrillar collagens consist of domains that are responsible for such processes as interaction with cells, binding of growth factors, and interaction with a number of structural proteins present in the extracellular matrix. A normal collagen consists of a sequence of domains that can be represented by a corresponding sequence of labels, e.g., D1D2D3D4. A collagenlike protein of the present invention contains regions of collagen II that contain multiples of a single domain (e.g., D1D1D1D1 or D4D4D4D4) chosen for its specific biological activity. By virtue of the multiplicity of the chosen domain, the density of sites having that specific biological activity is greater than it is in a normal collagen. A collagenlike protein according to this invention can thus be made to have properties that are necessary for tissue engineering.

  14. Protein tyrosine nitration

    PubMed Central

    Chaki, Mounira; Leterrier, Marina; Barroso, Juan B

    2009-01-01

    Nitric oxide metabolism in plant cells has a relative short history. Nitration is a chemical process which consists of introducing a nitro group (-NO2) into a chemical compound. in biological systems, this process has been found in different molecules such as proteins, lipids and nucleic acids that can affect its function. This mini-review offers an overview of this process with special emphasis on protein tyrosine nitration in plants and its involvement in the process of nitrosative stress. PMID:19826215

  15. The Malignant Protein Puzzle.

    PubMed

    Walker, Lary C; Jucker, Mathias

    2016-01-01

    When most people hear the words malignant and brain, cancer immediately comes to mind. But our authors argue that proteins can be malignant too, and can spread harmfully through the brain in neurodegenerative diseases that include Alzheimer's, Parkinson's, CTE, and ALS. Studying how proteins such as PrP, amyloid beta, tau, and others aggregate and spread, and kill brain cells, represents a crucial new frontier in neuroscience. PMID:27408676

  16. Bence-Jones protein - quantitative

    MedlinePlus

    Immunoglobulin light chains - urine; Urine Bence-Jones protein ... Bence-Jones proteins are a part of regular antibodies called light chains. These proteins are not normally in urine. Sometimes, when ...

  17. Disease specific protein corona

    NASA Astrophysics Data System (ADS)

    Rahman, M.; Mahmoudi, M.

    2015-03-01

    It is now well accepted that upon their entrance into the biological environments, the surface of nanomaterials would be covered by various biomacromolecules (e.g., proteins and lipids). The absorption of these biomolecules, so called `protein corona', onto the surface of (nano)biomaterials confers them a new `biological identity'. Although the formation of protein coronas on the surface of nanoparticles has been widely investigated, there are few reports on the effect of various diseases on the biological identity of nanoparticles. As the type of diseases may tremendously changes the composition of the protein source (e.g., human plasma/serum), one can expect that amount and composition of associated proteins in the corona composition may be varied, in disease type manner. Here, we show that corona coated silica and polystyrene nanoparticles (after interaction with in the plasma of the healthy individuals) could induce unfolding of fibrinogen, which promotes release of the inflammatory cytokines. However, no considerable releases of inflammatory cytokines were observed for corona coated graphene sheets. In contrast, the obtained corona coated silica and polystyrene nanoparticles from the hypofibrinogenemia patients could not induce inflammatory cytokine release where graphene sheets do. Therefore, one can expect that disease-specific protein coronas can provide a novel approach for applying nanomedicine to personalized medicine, improving diagnosis and treatment of different diseases tailored to the specific conditions and circumstances.

  18. Cardiolipin Interactions with Proteins.

    PubMed

    Planas-Iglesias, Joan; Dwarakanath, Himal; Mohammadyani, Dariush; Yanamala, Naveena; Kagan, Valerian E; Klein-Seetharaman, Judith

    2015-09-15

    Cardiolipins (CL) represent unique phospholipids of bacteria and eukaryotic mitochondria with four acyl chains and two phosphate groups that have been implicated in numerous functions from energy metabolism to apoptosis. Many proteins are known to interact with CL, and several cocrystal structures of protein-CL complexes exist. In this work, we describe the collection of the first systematic and, to the best of our knowledge, the comprehensive gold standard data set of all known CL-binding proteins. There are 62 proteins in this data set, 21 of which have nonredundant crystal structures with bound CL molecules available. Using binding patch analysis of amino acid frequencies, secondary structures and loop supersecondary structures considering phosphate and acyl chain binding regions together and separately, we gained a detailed understanding of the general structural and dynamic features involved in CL binding to proteins. Exhaustive docking of CL to all known structures of proteins experimentally shown to interact with CL demonstrated the validity of the docking approach, and provides a rich source of information for experimentalists who may wish to validate predictions.

  19. Cotton and Protein Interactions

    SciTech Connect

    Goheen, Steven C.; Edwards, J. V.; Rayburn, Alfred R.; Gaither, Kari A.; Castro, Nathan J.

    2006-06-30

    The adsorbent properties of important wound fluid proteins and cotton cellulose are reviewed. This review focuses on the adsorption of albumin to cotton-based wound dressings and some chemically modified derivatives targeted for chronic wounds. Adsorption of elastase in the presence of albumin was examined as a model to understand the interactive properties of these wound fluid components with cotton fibers. In the chronic non-healing wound, elastase appears to be over-expressed, and it digests tissue and growth factors, interfering with the normal healing process. Albumin is the most prevalent protein in wound fluid, and in highly to moderately exudative wounds, it may bind significantly to the fibers of wound dressings. Thus, the relative binding properties of both elastase and albumin to wound dressing fibers are of interest in the design of more effective wound dressings. The present work examines the binding of albumin to two different derivatives of cotton, and quantifies the elastase binding to the same derivatives following exposure of albumin to the fiber surface. An HPLC adsorption technique was employed coupled with a colorimetric enzyme assay to quantify the relative binding properties of albumin and elastase to cotton. The results of wound protein binding are discussed in relation to the porosity and surface chemistry interactions of cotton and wound proteins. Studies are directed to understanding the implications of protein adsorption phenomena in terms of fiber-protein models that have implications for rationally designing dressings for chronic wounds.

  20. Fast protein folding kinetics

    PubMed Central

    Gelman, Hannah; Gruebele, Martin

    2014-01-01

    Fast folding proteins have been a major focus of computational and experimental study because they are accessible to both techniques: they are small and fast enough to be reasonably simulated with current computational power, but have dynamics slow enough to be observed with specially developed experimental techniques. This coupled study of fast folding proteins has provided insight into the mechanisms which allow some proteins to find their native conformation well less than 1 ms and has uncovered examples of theoretically predicted phenomena such as downhill folding. The study of fast folders also informs our understanding of even “slow” folding processes: fast folders are small, relatively simple protein domains and the principles that govern their folding also govern the folding of more complex systems. This review summarizes the major theoretical and experimental techniques used to study fast folding proteins and provides an overview of the major findings of fast folding research. Finally, we examine the themes that have emerged from studying fast folders and briefly summarize their application to protein folding in general as well as some work that is left to do. PMID:24641816

  1. Membrane protein secretases.

    PubMed Central

    Hooper, N M; Karran, E H; Turner, A J

    1997-01-01

    A diverse range of membrane proteins of Type 1 or Type II topology also occur as a circulating, soluble form. These soluble forms are often derived from the membrane form by proteolysis by a group of enzymes referred to collectively as 'secretases' or 'sheddases'. The cleavage generally occurs close to the extracellular face of the membrane, releasing physiologically active protein. This secretion process also provides a mechanism for down-regulating the protein at the cell surface. Examples of such post-translational proteolysis are seen in the Alzheimer's amyloid precursor protein, the vasoregulatory enzyme angiotensin converting enzyme, transforming growth factor-alpha, the tumour necrosis factor ligand and receptor superfamilies, certain cytokine receptors, and others. Since the proteins concerned are involved in pathophysiological processes such as neurodegeneration, apoptosis, oncogenesis and inflammation, the secretases could provide novel therapeutic targets. Recent characterization of these individual secretases has revealed common features, particularly sensitivity to certain metalloprotease inhibitors and upregulation of activity by phorbol esters. It is therefore likely that a closely related family of metallosecretases controls the surface expression of multiple integral membrane proteins. Current knowledge of the various secretases are compared in this Review, and strategies for cell-free assays of such proteases are outlined as a prelude to their ultimate purification and cloning. PMID:9020855

  2. Cardiolipin Interactions with Proteins

    PubMed Central

    Planas-Iglesias, Joan; Dwarakanath, Himal; Mohammadyani, Dariush; Yanamala, Naveena; Kagan, Valerian E.; Klein-Seetharaman, Judith

    2015-01-01

    Cardiolipins (CL) represent unique phospholipids of bacteria and eukaryotic mitochondria with four acyl chains and two phosphate groups that have been implicated in numerous functions from energy metabolism to apoptosis. Many proteins are known to interact with CL, and several cocrystal structures of protein-CL complexes exist. In this work, we describe the collection of the first systematic and, to the best of our knowledge, the comprehensive gold standard data set of all known CL-binding proteins. There are 62 proteins in this data set, 21 of which have nonredundant crystal structures with bound CL molecules available. Using binding patch analysis of amino acid frequencies, secondary structures and loop supersecondary structures considering phosphate and acyl chain binding regions together and separately, we gained a detailed understanding of the general structural and dynamic features involved in CL binding to proteins. Exhaustive docking of CL to all known structures of proteins experimentally shown to interact with CL demonstrated the validity of the docking approach, and provides a rich source of information for experimentalists who may wish to validate predictions. PMID:26300339

  3. Multifunctional protein: cardiac ankyrin repeat protein*

    PubMed Central

    Zhang, Na; Xie, Xiao-jie; Wang, Jian-an

    2016-01-01

    Cardiac ankyrin repeat protein (CARP) not only serves as an important component of muscle sarcomere in the cytoplasm, but also acts as a transcription co-factor in the nucleus. Previous studies have demonstrated that CARP is up-regulated in some cardiovascular disorders and muscle diseases; however, its role in these diseases remains controversial now. In this review, we will discuss the continued progress in the research related to CARP, including its discovery, structure, and the role it plays in cardiac development and heart diseases. PMID:27143260

  4. Use of protein-protein interactions in affinity chromatography.

    PubMed

    Muronetz, V I; Sholukh, M; Korpela, T

    2001-10-30

    Biospecific recognition between proteins is a phenomenon that can be exploited for designing affinity-chromatographic purification systems for proteins. In principle, the approach is straightforward, and there are usually many alternative ways, since a protein can be always found which binds specifically enough to the desired protein. Routine immunoaffinity chromatography utilizes the recognition of antigenic epitopes by antibodies. However, forces involved in protein-protein interactions as well the forces keeping the three-dimensional structures of proteins intact are complicated, and proteins are easily unfolded by various factors with unpredictable results. Because of this and because of the generally high association strength between proteins, the correct adjustment of binding forces between an immobilized protein and the protein to be purified as well as the release of bound proteins in biologically active form from affinity complexes are the main problem. Affinity systems involving interactions like enzyme-enzyme, subunit-oligomer, protein-antibody, protein-chaperone and the specific features involved in each case are presented as examples. This article also aims to sketch prospects for further development of the use of protein-protein interactions for the purification of proteins. PMID:11694271

  5. Protein crystal growth in space

    NASA Technical Reports Server (NTRS)

    Bugg, C. E.; Clifford, D. W.

    1987-01-01

    The advantages of protein crystallization in space, and the applications of protein crystallography to drug design, protein engineering, and the design of synthetic vaccines are examined. The steps involved in using protein crystallography to determine the three-dimensional structure of a protein are discussed. The growth chamber design and the hand-held apparatus developed for protein crystal growth by vapor diffusion techniques (hanging-drop method) are described; the experimental data from the four Shuttle missions are utilized to develop hardware for protein crystal growth in space and to evaluate the effects of gravity on protein crystal growth.

  6. Modeling Mercury in Proteins

    SciTech Connect

    Smith, Jeremy C; Parks, Jerry M

    2016-01-01

    Mercury (Hg) is a naturally occurring element that is released into the biosphere both by natural processes and anthropogenic activities. Although its reduced, elemental form Hg(0) is relatively non-toxic, other forms such as Hg2+ and, in particular, its methylated form, methylmercury, are toxic, with deleterious effects on both ecosystems and humans. Microorganisms play important roles in the transformation of mercury in the environment. Inorganic Hg2+ can be methylated by certain bacteria and archaea to form methylmercury. Conversely, bacteria also demethylate methylmercury and reduce Hg2+ to relatively inert Hg(0). Transformations and toxicity occur as a result of mercury interacting with various proteins. Clearly, then, understanding the toxic effects of mercury and its cycling in the environment requires characterization of these interactions. Computational approaches are ideally suited to studies of mercury in proteins because they can provide a detailed picture and circumvent issues associated with toxicity. Here we describe computational methods for investigating and characterizing how mercury binds to proteins, how inter- and intra-protein transfer of mercury is orchestrated in biological systems, and how chemical reactions in proteins transform the metal. We describe quantum chemical analyses of aqueous Hg(II), which reveal critical factors that determine ligand binding propensities. We then provide a perspective on how we used chemical reasoning to discover how microorganisms methylate mercury. We also highlight our combined computational and experimental studies of the proteins and enzymes of the mer operon, a suite of genes that confers mercury resistance in many bacteria. Lastly, we place work on mercury in proteins in the context of what is needed for a comprehensive multi-scale model of environmental mercury cycling.

  7. Bioinformatics and Moonlighting Proteins.

    PubMed

    Hernández, Sergio; Franco, Luís; Calvo, Alejandra; Ferragut, Gabriela; Hermoso, Antoni; Amela, Isaac; Gómez, Antonio; Querol, Enrique; Cedano, Juan

    2015-01-01

    Multitasking or moonlighting is the capability of some proteins to execute two or more biochemical functions. Usually, moonlighting proteins are experimentally revealed by serendipity. For this reason, it would be helpful that Bioinformatics could predict this multifunctionality, especially because of the large amounts of sequences from genome projects. In the present work, we analyze and describe several approaches that use sequences, structures, interactomics, and current bioinformatics algorithms and programs to try to overcome this problem. Among these approaches are (a) remote homology searches using Psi-Blast, (b) detection of functional motifs and domains, (c) analysis of data from protein-protein interaction databases (PPIs), (d) match the query protein sequence to 3D databases (i.e., algorithms as PISITE), and (e) mutation correlation analysis between amino acids by algorithms as MISTIC. Programs designed to identify functional motif/domains detect mainly the canonical function but usually fail in the detection of the moonlighting one, Pfam and ProDom being the best methods. Remote homology search by Psi-Blast combined with data from interactomics databases (PPIs) has the best performance. Structural information and mutation correlation analysis can help us to map the functional sites. Mutation correlation analysis can only be used in very specific situations - it requires the existence of multialigned family protein sequences - but can suggest how the evolutionary process of second function acquisition took place. The multitasking protein database MultitaskProtDB (http://wallace.uab.es/multitask/), previously published by our group, has been used as a benchmark for the all of the analyses. PMID:26157797

  8. The Hedgehog protein family.

    PubMed

    Bürglin, Thomas R

    2008-01-01

    The Hedgehog (Hh) pathway is one of the fundamental signal transduction pathways in animal development and is also involved in stem-cell maintenance and carcinogenesis. The hedgehog (hh) gene was first discovered in Drosophila, and members of the family have since been found in most metazoa. Hh proteins are composed of two domains, an amino-terminal domain HhN, which has the biological signal activity, and a carboxy-terminal autocatalytic domain HhC, which cleaves Hh into two parts in an intramolecular reaction and adds a cholesterol moiety to HhN. HhC has sequence similarity to the self-splicing inteins, and the shared region is termed Hint. New classes of proteins containing the Hint domain have been discovered recently in bacteria and eukaryotes, and the Hog class, of which Hh proteins comprise one family, is widespread throughout eukaryotes. The non-Hh Hog proteins have carboxy-terminal domains (the Hog domain) highly similar to HhC, although they lack the HhN domain, and instead have other amino-terminal domains. Hog proteins are found in many protists, but the Hh family emerged only in early metazoan evolution. HhN is modified by cholesterol at its carboxyl terminus and by palmitate at its amino terminus in both flies and mammals. The modified HhN is released from the cell and travels through the extracellular space. On binding its receptor Patched, it relieves the inhibition that Patched exerts on Smoothened, a G-protein-coupled receptor. The resulting signaling cascade converges on the transcription factor Cubitus interruptus (Ci), or its mammalian counterparts, the Gli proteins, which activate or repress target genes.

  9. Self-Assembling Protein Microarrays

    NASA Astrophysics Data System (ADS)

    Ramachandran, Niroshan; Hainsworth, Eugenie; Bhullar, Bhupinder; Eisenstein, Samuel; Rosen, Benjamin; Lau, Albert Y.; C. Walter, Johannes; LaBaer, Joshua

    2004-07-01

    Protein microarrays provide a powerful tool for the study of protein function. However, they are not widely used, in part because of the challenges in producing proteins to spot on the arrays. We generated protein microarrays by printing complementary DNAs onto glass slides and then translating target proteins with mammalian reticulocyte lysate. Epitope tags fused to the proteins allowed them to be immobilized in situ. This obviated the need to purify proteins, avoided protein stability problems during storage, and captured sufficient protein for functional studies. We used the technology to map pairwise interactions among 29 human DNA replication initiation proteins, recapitulate the regulation of Cdt1 binding to select replication proteins, and map its geminin-binding domain.

  10. Purine inhibitors of protein kinases, G proteins and polymerases

    DOEpatents

    Gray, Nathanael S.; Schultz, Peter; Kim, Sung-Hou; Meijer, Laurent

    2001-07-03

    The present invention relates to purine analogs that inhibit, inter alia, protein kinases, G-proteins and polymerases. In addition, the present invention relates to methods of using such purine analogs to inhibit protein kinases, G-proteins, polymerases and other cellular processes and to treat cellular proliferative diseases.

  11. Benchtop Detection of Proteins

    NASA Technical Reports Server (NTRS)

    Scardelletti, Maximilian C.; Varaljay, Vanessa

    2007-01-01

    A process, and a benchtop-scale apparatus for implementing the process, have been developed to detect proteins associated with specific microbes in water. The process and apparatus may also be useful for detection of proteins in other, more complex liquids. There may be numerous potential applications, including monitoring lakes and streams for contamination, testing of blood and other bodily fluids in medical laboratories, and testing for microbial contamination of liquids in restaurants and industrial food-processing facilities. A sample can be prepared and analyzed by use of this process and apparatus within minutes, whereas an equivalent analysis performed by use of other processes and equipment can often take hours to days. The process begins with the conjugation of near-infrared-fluorescent dyes to antibodies that are specific to a particular protein. Initially, the research has focused on using near-infrared dyes to detect antigens or associated proteins in solution, which has proven successful vs. microbial cells, and streamlining the technique in use for surface protein detection on microbes would theoretically render similar results. However, it is noted that additional work is needed to transition protein-based techniques to microbial cell detection. Consequently, multiple such dye/antibody pairs could be prepared to enable detection of multiple selected microbial species, using a different dye for each species. When excited by near-infrared light of a suitable wavelength, each dye fluoresces at a unique longer wavelength that differs from those of the other dyes, enabling discrimination among the various species. In initial tests, the dye/antibody pairs are mixed into a solution suspected of containing the selected proteins, causing the binding of the dye/antibody pairs to such suspect proteins that may be present. The solution is then run through a microcentrifuge that includes a membrane that acts as a filter in that it retains the dye/antibody/protein

  12. Heat Capacity in Proteins

    NASA Astrophysics Data System (ADS)

    Prabhu, Ninad V.; Sharp, Kim A.

    2005-05-01

    Heat capacity (Cp) is one of several major thermodynamic quantities commonly measured in proteins. With more than half a dozen definitions, it is the hardest of these quantities to understand in physical terms, but the richest in insight. There are many ramifications of observed Cp changes: The sign distinguishes apolar from polar solvation. It imparts a temperature (T) dependence to entropy and enthalpy that may change their signs and which of them dominate. Protein unfolding usually has a positive ΔCp, producing a maximum in stability and sometimes cold denaturation. There are two heat capacity contributions, from hydration and protein-protein interactions; which dominates in folding and binding is an open question. Theoretical work to date has dealt mostly with the hydration term and can account, at least semiquantitatively, for the major Cp-related features: the positive and negative Cp of hydration for apolar and polar groups, respectively; the convergence of apolar group hydration entropy at T ≈ 112°C; the decrease in apolar hydration Cp with increasing T; and the T-maximum in protein stability and cold denaturation.

  13. Electrochemical nanomoulding through proteins

    NASA Astrophysics Data System (ADS)

    Allred, Daniel B.

    The continued improvements in performance of modern electronic devices are directly related to the manufacturing of smaller, denser features on surfaces. Electrochemical fabrication has played a large role in continuing this trend due to its low cost and ease of scaleability toward ever smaller dimensions. This work introduces the concept of using proteins, essentially monodisperse complex polymers whose three-dimensional structures are fixed by their encoded amino acid sequences, as "moulds" around which nanostructures can be built by electrochemical fabrication. Bacterial cell-surface layer proteins, or "S-layer" proteins, from two organisms---Deinococcus radiodurans and Sporosarcina ureae---were used as the "moulds" for electrochemical fabrication. The proteins are easily purified as micron-sized sheets of periodic molecular complexes with 18-nm hexagonal and 13-nm square unit cell lattices, respectively. Direct imaging by transmission electron microscopy on ultrathin noble metal films without sample preparation eliminates potential artifacts to the high surface energy substrates necessary for high nucleation densities. Characterization involved imaging, electron diffraction, spectroscopy, and three-dimensional reconstruction. The S-layer protein of D. radiodurans was further subjected to an atomic force microscope based assay to determine the integrity of its structure and long-range order and was found to be useful for fabrication from around pH 3 to 12.

  14. Nanophotonics of protein amyloids

    NASA Astrophysics Data System (ADS)

    Bhattacharya, Mily; Mukhopadhyay, Samrat

    2014-04-01

    Technological breakthroughs in the super-resolution optical imaging techniques have enriched our current understanding of a range of biological systems and biomolecular processes at the nanoscopic spatial resolution. Protein amyloids are an important class of ordered protein assemblies consisting of misfolded proteins that are implicated in a wide range of devastating human diseases. In order to decipher the structural basis of the supramolecular protein assembly in amyloids and their detrimental interactions with the cell membranes, it is important to employ high-resolution optical imaging techniques. Additionally, amyloids could serve as novel biological nanomaterials for a variety of potential applications. In this review, we summarize a few examples of the utility of near-field scanning optical imaging methodologies to obtain a wealth of structural information into the nanoscale amyloid assembly. Although the near-field technologies were developed several decades ago, it is only recently that these methodologies are being applied and adapted for amyloid research to yield novel information pertaining to the exciting nanoscopic world of protein aggregates. We believe that the account on the nanophotonics of amyloids described in this review will be useful for the future studies on the biophysics of amyloids.

  15. Protein Crowding Is a Determinant of Lipid Droplet Protein Composition.

    PubMed

    Kory, Nora; Thiam, Abdou-Rachid; Farese, Robert V; Walther, Tobias C

    2015-08-10

    Lipid droplets (LDs) are lipid storage organelles that grow or shrink, depending on the availability of metabolic energy. Proteins recruited to LDs mediate many metabolic functions, including phosphatidylcholine and triglyceride synthesis. How the LD protein composition is tuned to the supply and demand for lipids remains unclear. We show that LDs, in contrast to other organelles, have limited capacity for protein binding. Consequently, macromolecular crowding plays a major role in determining LD protein composition. During lipolysis, when LDs and their surfaces shrink, some, but not all, proteins become displaced. In vitro studies show that macromolecular crowding, rather than changes in monolayer lipid composition, causes proteins to fall off the LD surface. As predicted by a crowding model, proteins compete for binding to the surfaces of LDs. Moreover, the LD binding affinity determines protein localization during lipolysis. Our findings identify protein crowding as an important principle in determining LD protein composition. PMID:26212136

  16. The detection of DNA-binding proteins by protein blotting.

    PubMed Central

    Bowen, B; Steinberg, J; Laemmli, U K; Weintraub, H

    1980-01-01

    A method, called "protein blotting," for the detection of DNA-binding proteins is described. Proteins are separated on an SDA-polyacrylamide gel. The gel is sandwiched between 2 nitrocellulose filters and the proteins allowed to diffuse out of the gel and onto the filters. The proteins are tightly bound to each filter, producing a replica of the original gel pattern. The replica is used to detect DNA-binding proteins, RNA-binding proteins or histone-binding proteins by incubation of the filter with [32P]DNA, [125I]RNA, or [125I] histone. Evidence is also presented that specific protein-DNA interactions may be detected by this technique; under appropriate conditions, the lac repressor binds only to DNA containing the lac operator. Strategies for the detection of specific protein-DNA interactions are discussed. Images PMID:6243775

  17. Plant protein kinase substrates identification using protein microarrays.

    PubMed

    Ma, Shisong; Dinesh-Kumar, Savithramma P

    2015-01-01

    Protein kinases regulate signaling pathways by phosphorylating their targets. They play critical roles in plant signaling networks. Although many important protein kinases have been identified in plants, their substrates are largely unknown. We have developed and produced plant protein microarrays with more than 15,000 purified plant proteins. Here, we describe a detailed protocol to use these microarrays to identify plant protein kinase substrates via in vitro phosphorylation assays on these arrays. PMID:25930701

  18. Advanced protein formulations.

    PubMed

    Wang, Wei

    2015-07-01

    It is well recognized that protein product development is far more challenging than that for small-molecule drugs. The major challenges include inherent sensitivity to different types of stresses during the drug product manufacturing process, high rate of physical and chemical degradation during long-term storage, and enhanced aggregation and/or viscosity at high protein concentrations. In the past decade, many novel formulation concepts and technologies have been or are being developed to address these product development challenges for proteins. These concepts and technologies include use of uncommon/combination of formulation stabilizers, conjugation or fusion with potential stabilizers, site-specific mutagenesis, and preparation of nontraditional types of dosage forms-semiaqueous solutions, nonfreeze-dried solid formulations, suspensions, and other emerging concepts. No one technology appears to be mature, ideal, and/or adequate to address all the challenges. These gaps will likely remain in the foreseeable future and need significant efforts for ultimate resolution.

  19. Protein Crystal Serum Albumin

    NASA Technical Reports Server (NTRS)

    1998-01-01

    As the most abundant protein in the circulatory system albumin contributes 80% to colloid osmotic blood pressure. Albumin is also chiefly responsible for the maintenance of blood pH. It is located in every tissue and bodily secretion, with extracellular protein comprising 60% of total albumin. Perhaps the most outstanding property of albumin is its ability to bind reversibly to an incredible variety of ligands. It is widely accepted in the pharmaceutical industry that the overall distribution, metabolism, and efficiency of many drugs are rendered ineffective because of their unusually high affinity for this abundant protein. An understanding of the chemistry of the various classes of pharmaceutical interactions with albumin can suggest new approaches to drug therapy and design. Principal Investigator: Dan Carter/New Century Pharmaceuticals

  20. Targeted antithrombotic protein micelles.

    PubMed

    Kim, Wookhyun; Haller, Carolyn; Dai, Erbin; Wang, Xiowei; Hagemeyer, Christoph E; Liu, David R; Peter, Karlheinz; Chaikof, Elliot L

    2015-01-26

    Activated platelets provide a promising target for imaging inflammatory and thrombotic events along with site-specific delivery of a variety of therapeutic agents. Multifunctional protein micelles bearing targeting and therapeutic proteins were now obtained by one-pot transpeptidation using an evolved sortase A. Conjugation to the corona of a single-chain antibody (scFv), which binds to the ligand-induced binding site (LIBS) of activated GPIIb/IIIa receptors, enabled the efficient detection of thrombi. The inhibition of thrombus formation was subsequently accomplished by incorporating the catalytically active domain of thrombomodulin (TM) onto the micelle corona for the local generation of activated protein C, which inhibits the formation of thrombin. An effective strategy has been developed for the preparation of protein micelles that can be targeted to sites of activated platelets with broad potential for treatment of acute thrombotic events. PMID:25504546

  1. Protein crystallization studies

    NASA Technical Reports Server (NTRS)

    Lyne, James Evans

    1996-01-01

    The Structural Biology laboratory at NASA Marshall Spaceflight Center uses x-ray crystallographic techniques to conduct research into the three-dimensional structure of a wide variety of proteins. A major effort in the laboratory involves an ongoing study of human serum albumin (the principal protein in human plasma) and its interaction with various endogenous substances and pharmaceutical agents. Another focus is on antigenic and functional proteins from several pathogenic organisms including the human immunodeficiency virus (HIV) and the widespread parasitic genus, Schistosoma. My efforts this summer have been twofold: first, to identify clinically significant drug interactions involving albumin binding displacement and to initiate studies of the three-dimensional structure of albumin complexed with these agents, and secondly, to establish collaborative efforts to extend the lab's work on human pathogens.

  2. Advanced protein formulations

    PubMed Central

    Wang, Wei

    2015-01-01

    It is well recognized that protein product development is far more challenging than that for small-molecule drugs. The major challenges include inherent sensitivity to different types of stresses during the drug product manufacturing process, high rate of physical and chemical degradation during long-term storage, and enhanced aggregation and/or viscosity at high protein concentrations. In the past decade, many novel formulation concepts and technologies have been or are being developed to address these product development challenges for proteins. These concepts and technologies include use of uncommon/combination of formulation stabilizers, conjugation or fusion with potential stabilizers, site-specific mutagenesis, and preparation of nontraditional types of dosage forms—semiaqueous solutions, nonfreeze-dried solid formulations, suspensions, and other emerging concepts. No one technology appears to be mature, ideal, and/or adequate to address all the challenges. These gaps will likely remain in the foreseeable future and need significant efforts for ultimate resolution. PMID:25858529

  3. Thermodynamics of Protein Aggregation

    NASA Astrophysics Data System (ADS)

    Osborne, Kenneth L.; Barz, Bogdan; Bachmann, Michael; Strodel, Birgit

    Amyloid protein aggregation characterizes many neurodegenerative disorders, including Alzheimer's, Parkinson's, and Creutz- feldt-Jakob disease. Evidence suggests that amyloid aggregates may share similar aggregation pathways, implying simulation of full-length amyloid proteins is not necessary for understanding amyloid formation. In this study we simulate GNNQQNY, the N-terminal prion-determining domain of the yeast protein Sup35 to investigate the thermodynamics of structural transitions during aggregation. We use a coarse-grained model with replica-exchange molecular dynamics to investigate the association of 3-, 6-, and 12-chain GNNQQNY systems and we determine the aggregation pathway by studying aggregation states of GN- NQQNY. We find that the aggregation of the hydrophilic GNNQQNY sequence is mainly driven by H-bond formation, leading to the formation of /3-sheets from the very beginning of the assembly process. Condensation (aggregation) and ordering take place simultaneously, which is underpinned by the occurrence of a single heat capacity peak only.

  4. How to Study Protein-protein Interactions.

    PubMed

    Podobnik, Marjetka; Kraševec, Nada; Bedina Zavec, Apolonija; Naneh, Omar; Flašker, Ajda; Caserman, Simon; Hodnik, Vesna; Anderluh, Gregor

    2016-01-01

    Physical and functional interactions between molecules in living systems are central to all biological processes. Identification of protein complexes therefore is becoming increasingly important to gain a molecular understanding of cells and organisms. Several powerful methodologies and techniques have been developed to study molecular interactions and thus help elucidate their nature and role in biology as well as potential ways how to interfere with them. All different techniques used in these studies have their strengths and weaknesses and since they are mostly employed in in vitro conditions, a single approach can hardly accurately reproduce interactions that happen under physiological conditions. However, complementary usage of as many as possible available techniques can lead to relatively realistic picture of the biological process. Here we describe several proteomic, biophysical and structural tools that help us understand the nature and mechanism of these interactions. PMID:27640371

  5. Discovery of binding proteins for a protein target using protein-protein docking-based virtual screening.

    PubMed

    Zhang, Changsheng; Tang, Bo; Wang, Qian; Lai, Luhua

    2014-10-01

    Target structure-based virtual screening, which employs protein-small molecule docking to identify potential ligands, has been widely used in small-molecule drug discovery. In the present study, we used a protein-protein docking program to identify proteins that bind to a specific target protein. In the testing phase, an all-to-all protein-protein docking run on a large dataset was performed. The three-dimensional rigid docking program SDOCK was used to examine protein-protein docking on all protein pairs in the dataset. Both the binding affinity and features of the binding energy landscape were considered in the scoring function in order to distinguish positive binding pairs from negative binding pairs. Thus, the lowest docking score, the average Z-score, and convergency of the low-score solutions were incorporated in the analysis. The hybrid scoring function was optimized in the all-to-all docking test. The docking method and the hybrid scoring function were then used to screen for proteins that bind to tumor necrosis factor-α (TNFα), which is a well-known therapeutic target for rheumatoid arthritis and other autoimmune diseases. A protein library containing 677 proteins was used for the screen. Proteins with scores among the top 20% were further examined. Sixteen proteins from the top-ranking 67 proteins were selected for experimental study. Two of these proteins showed significant binding to TNFα in an in vitro binding study. The results of the present study demonstrate the power and potential application of protein-protein docking for the discovery of novel binding proteins for specific protein targets.

  6. Modeling Mercury in Proteins.

    PubMed

    Parks, J M; Smith, J C

    2016-01-01

    Mercury (Hg) is a naturally occurring element that is released into the biosphere both by natural processes and anthropogenic activities. Although its reduced, elemental form Hg(0) is relatively nontoxic, other forms such as Hg(2+) and, in particular, its methylated form, methylmercury, are toxic, with deleterious effects on both ecosystems and humans. Microorganisms play important roles in the transformation of mercury in the environment. Inorganic Hg(2+) can be methylated by certain bacteria and archaea to form methylmercury. Conversely, bacteria also demethylate methylmercury and reduce Hg(2+) to relatively inert Hg(0). Transformations and toxicity occur as a result of mercury interacting with various proteins. Clearly, then, understanding the toxic effects of mercury and its cycling in the environment requires characterization of these interactions. Computational approaches are ideally suited to studies of mercury in proteins because they can provide a detailed molecular picture and circumvent issues associated with toxicity. Here, we describe computational methods for investigating and characterizing how mercury binds to proteins, how inter- and intraprotein transfer of mercury is orchestrated in biological systems, and how chemical reactions in proteins transform the metal. We describe quantum chemical analyses of aqueous Hg(II), which reveal critical factors that determine ligand-binding propensities. We then provide a perspective on how we used chemical reasoning to discover how microorganisms methylate mercury. We also highlight our combined computational and experimental studies of the proteins and enzymes of the mer operon, a suite of genes that confer mercury resistance in many bacteria. Lastly, we place work on mercury in proteins in the context of what is needed for a comprehensive multiscale model of environmental mercury cycling.

  7. Modeling Mercury in Proteins.

    PubMed

    Parks, J M; Smith, J C

    2016-01-01

    Mercury (Hg) is a naturally occurring element that is released into the biosphere both by natural processes and anthropogenic activities. Although its reduced, elemental form Hg(0) is relatively nontoxic, other forms such as Hg(2+) and, in particular, its methylated form, methylmercury, are toxic, with deleterious effects on both ecosystems and humans. Microorganisms play important roles in the transformation of mercury in the environment. Inorganic Hg(2+) can be methylated by certain bacteria and archaea to form methylmercury. Conversely, bacteria also demethylate methylmercury and reduce Hg(2+) to relatively inert Hg(0). Transformations and toxicity occur as a result of mercury interacting with various proteins. Clearly, then, understanding the toxic effects of mercury and its cycling in the environment requires characterization of these interactions. Computational approaches are ideally suited to studies of mercury in proteins because they can provide a detailed molecular picture and circumvent issues associated with toxicity. Here, we describe computational methods for investigating and characterizing how mercury binds to proteins, how inter- and intraprotein transfer of mercury is orchestrated in biological systems, and how chemical reactions in proteins transform the metal. We describe quantum chemical analyses of aqueous Hg(II), which reveal critical factors that determine ligand-binding propensities. We then provide a perspective on how we used chemical reasoning to discover how microorganisms methylate mercury. We also highlight our combined computational and experimental studies of the proteins and enzymes of the mer operon, a suite of genes that confer mercury resistance in many bacteria. Lastly, we place work on mercury in proteins in the context of what is needed for a comprehensive multiscale model of environmental mercury cycling. PMID:27497164

  8. FLOW BEHAVIOR OF PROTEIN BLENDS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Blending proteins can increase textural strength or enhance taste or mouth feel, such as blending soy with whey to improve taste. In this study, we measured the viscosity of various combinations of six proteins (whey protein isolates, calcium caseinate, soy protein isolates, wheat gluten, egg album...

  9. DELIVERY OF THERAPEUTIC PROTEINS

    PubMed Central

    Pisal, Dipak S.; Kosloski, Matthew P.; Balu-Iyer, Sathy V.

    2009-01-01

    The safety and efficacy of protein therapeutics are limited by three interrelated pharmaceutical issues, in vitro and in vivo instability, immunogenicity and shorter half-lives. Novel drug modifications for overcoming these issues are under investigation and include covalent attachment of poly(ethylene glycol) (PEG), polysialic acid, or glycolic acid, as well as developing new formulations containing nanoparticulate or colloidal systems (e.g. liposomes, polymeric microspheres, polymeric nanoparticles). Such strategies have the potential to develop as next generation protein therapeutics. This review includes a general discussion on these delivery approaches. PMID:20049941

  10. Kinetics of protein aggregation

    NASA Astrophysics Data System (ADS)

    Knowles, Tuomas

    2015-03-01

    Aggregation into linear nanostructures, notably amyloid and amyloid-like fibrils, is a common form of behaviour exhibited by a range of peptides and proteins. This process was initially discovered in the context of the aetiology of a range of neurodegenerative diseases, but has recently been recognised to of general significance and has been found at the origin of a number of beneficial functional roles in nature, including as catalytic scaffolds and functional components in biofilms. This talk discusses our ongoing efforts to study the kinetics of linear protein self-assembly by using master equation approaches combined with global analysis of experimental data.

  11. Lipid-transfer proteins.

    PubMed

    Ng, Tzi Bun; Cheung, Randy Chi Fai; Wong, Jack Ho; Ye, Xiujuan

    2012-01-01

    Lipid-transfer proteins (LTPs) are basic proteins found in abundance in higher plants. LTPs play lots of roles in plants such as participation in cutin formation, embryogenesis, defense reactions against phytopathogens, symbiosis, and the adaptation of plants to various environmental conditions. In addition, LTPs from field mustard and Chinese daffodil exhibit antiproliferative activity against human cancer cells. LTPs from chili pepper and coffee manifest inhibitory activity against fungi pathogenic to humans such as Candida species. The intent of this article is to review LTPs in the plant kingdom. PMID:23193591

  12. Coupled transport protein systems.

    PubMed

    Thatcher, Jack D

    2013-04-16

    This set of animated lessons provides examples of how transport proteins interact in coupled systems to produce physiologic effects. The gastric pumps animation depicts the secretion of hydrochloric acid into the gastric lumen. The animation called glucose absorption depicts glucose absorption by intestinal epithelial cells. The CFTR animation explains how the cystic fibrosis conductance transmembrane regulator (CFTR) functions as a key component of a coupled system of transport proteins that clears the pulmonary system of mucus and inhaled particulates. These animations serve as valuable resources for any collegiate-level course that describes these processes. Courses that might use them include introductory biology, biochemistry, biophysics, cell biology, pharmacology, and physiology.

  13. Protein production and purification

    PubMed Central

    2010-01-01

    In selecting a method to produce a recombinant protein, a researcher is faced with a bewildering array of choices as to where to start. To facilitate decision-making, we describe a consensus ‘what to try first’ strategy based on our collective analysis of the expression and purification of over 10,000 different proteins. This review presents methods that could be applied at the outset of any project, a prioritized list of alternate strategies and a list of pitfalls that trip many new investigators. PMID:18235434

  14. Protein energy malnutrition.

    PubMed

    Grover, Zubin; Ee, Looi C

    2009-10-01

    Protein energy malnutrition (PEM) is a common problem worldwide and occurs in both developing and industrialized nations. In the developing world, it is frequently a result of socioeconomic, political, or environmental factors. In contrast, protein energy malnutrition in the developed world usually occurs in the context of chronic disease. There remains much variation in the criteria used to define malnutrition, with each method having its own limitations. Early recognition, prompt management, and robust follow up are critical for best outcomes in preventing and treating PEM.

  15. Late embryogenesis abundant proteins

    PubMed Central

    Olvera-Carrillo, Yadira; Reyes, José Luis

    2011-01-01

    Late Embryogenesis Abundant (LEA) proteins accumulate at the onset of seed desiccation and in response to water deficit in vegetative plant tissues. The typical LEA proteins are highly hydrophilic and intrinsically unstructured. They have been classified in different families, each one showing distinctive conserved motifs. In this manuscript we present and discuss some of the recent findings regarding their role in plant adaptation to water deficit, as well as those concerning to their possible function, and how it can be related to their intrinsic structural flexibility. PMID:21447997

  16. Protein Bodies of the Soybean

    PubMed Central

    Tombs, M. P.

    1967-01-01

    Some microscope observations of the protein bodies of the cotyledon cells of the soybean (Glycine max) are described, together with changes in their appearance which occur on germination. Density gradient centrifugation permits the isolation of protein bodies from soymeal. They contain about 70% of the protein of the bean. Only 1 protein could be detected in them: glycinin, the major soybean protein. The protein bodies were fractionated to light and heavy fractions. The former contained 97.5% protein, the latter 78.5%. RNA, phytic acid and lipids were also present. The 2 fractions probably differ only in the extent of contamination by other cell fragments. Images PMID:16656574

  17. NextGen protein design

    PubMed Central

    Regan, Lynne

    2014-01-01

    Protein engineering is at an exciting stage because designed protein–protein interactions are being used in many applications. For instance, three designed proteins are now in clinical trials. Although there have been many successes over the last decade, protein engineering still faces numerous challenges. Often, designs do not work as anticipated and they still require substantial redesign. The present review focuses on the successes, the challenges and the limitations of rational protein design today. PMID:24059497

  18. Accessory proteins for heterotrimeric G-proteins in the kidney

    PubMed Central

    Park, Frank

    2015-01-01

    Heterotrimeric G-proteins play a fundamentally important role in regulating signal transduction pathways in the kidney. Accessory proteins are being identified as direct binding partners for heterotrimeric G-protein α or βγ subunits to promote more diverse mechanisms by which G-protein signaling is controlled. In some instances, accessory proteins can modulate the signaling magnitude, localization, and duration following the activation of cell membrane-associated receptors. Alternatively, accessory proteins complexed with their G-protein α or βγ subunits can promote non-canonical models of signaling activity within the cell. In this review, we will highlight the expression profile, localization and functional importance of these newly identified accessory proteins to control the function of select G-protein subunits under normal and various disease conditions observed in the kidney. PMID:26300785

  19. Protein-protein interactions: methods for detection and analysis.

    PubMed Central

    Phizicky, E M; Fields, S

    1995-01-01

    The function and activity of a protein are often modulated by other proteins with which it interacts. This review is intended as a practical guide to the analysis of such protein-protein interactions. We discuss biochemical methods such as protein affinity chromatography, affinity blotting, coimmunoprecipitation, and cross-linking; molecular biological methods such as protein probing, the two-hybrid system, and phage display: and genetic methods such as the isolation of extragenic suppressors, synthetic mutants, and unlinked noncomplementing mutants. We next describe how binding affinities can be evaluated by techniques including protein affinity chromatography, sedimentation, gel filtration, fluorescence methods, solid-phase sampling of equilibrium solutions, and surface plasmon resonance. Finally, three examples of well-characterized domains involved in multiple protein-protein interactions are examined. The emphasis of the discussion is on variations in the approaches, concerns in evaluating the results, and advantages and disadvantages of the techniques. PMID:7708014

  20. Protein Molecular Structures, Protein SubFractions, and Protein Availability Affected by Heat Processing: A Review

    SciTech Connect

    Yu,P.

    2007-01-01

    The utilization and availability of protein depended on the types of protein and their specific susceptibility to enzymatic hydrolysis (inhibitory activities) in the gastrointestine and was highly associated with protein molecular structures. Studying internal protein structure and protein subfraction profiles leaded to an understanding of the components that make up a whole protein. An understanding of the molecular structure of the whole protein was often vital to understanding its digestive behavior and nutritive value in animals. In this review, recently obtained information on protein molecular structural effects of heat processing was reviewed, in relation to protein characteristics affecting digestive behavior and nutrient utilization and availability. The emphasis of this review was on (1) using the newly advanced synchrotron technology (S-FTIR) as a novel approach to reveal protein molecular chemistry affected by heat processing within intact plant tissues; (2) revealing the effects of heat processing on the profile changes of protein subfractions associated with digestive behaviors and kinetics manipulated by heat processing; (3) prediction of the changes of protein availability and supply after heat processing, using the advanced DVE/OEB and NRC-2001 models, and (4) obtaining information on optimal processing conditions of protein as intestinal protein source to achieve target values for potential high net absorbable protein in the small intestine. The information described in this article may give better insight in the mechanisms involved and the intrinsic protein molecular structural changes occurring upon processing.

  1. Regulators of G-protein-signaling proteins: negative modulators of G-protein-coupled receptor signaling.

    PubMed

    Woodard, Geoffrey E; Jardín, Isaac; Berna-Erro, A; Salido, Gines M; Rosado, Juan A

    2015-01-01

    Regulators of G-protein-signaling (RGS) proteins are a category of intracellular proteins that have an inhibitory effect on the intracellular signaling produced by G-protein-coupled receptors (GPCRs). RGS along with RGS-like proteins switch on through direct contact G-alpha subunits providing a variety of intracellular functions through intracellular signaling. RGS proteins have a common RGS domain that binds to G alpha. RGS proteins accelerate GTPase and thus enhance guanosine triphosphate hydrolysis through the alpha subunit of heterotrimeric G proteins. As a result, they inactivate the G protein and quickly turn off GPCR signaling thus terminating the resulting downstream signals. Activity and subcellular localization of RGS proteins can be changed through covalent molecular changes to the enzyme, differential gene splicing, and processing of the protein. Other roles of RGS proteins have shown them to not be solely committed to being inhibitors but behave more as modulators and integrators of signaling. RGS proteins modulate the duration and kinetics of slow calcium oscillations and rapid phototransduction and ion signaling events. In other cases, RGS proteins integrate G proteins with signaling pathways linked to such diverse cellular responses as cell growth and differentiation, cell motility, and intracellular trafficking. Human and animal studies have revealed that RGS proteins play a vital role in physiology and can be ideal targets for diseases such as those related to addiction where receptor signaling seems continuously switched on.

  2. Protein Crystal Bovine Insulin

    NASA Technical Reports Server (NTRS)

    1991-01-01

    The comparison of protein crystal, Bovine Insulin space-grown (left) and earth-grown (right). Facilitates the incorporation of glucose into cells. In diabetics, there is either a decrease in or complete lack of insulin, thereby leading to several harmful complications. Principal Investigator is Larry DeLucas.

  3. Protein nano-crystallogenesis.

    PubMed

    Kuil, Maxim E; Bodenstaff, E Rene; Hoedemaeker, Flip J; Abrahams, Jan Pieter

    2002-03-13

    We demonstrate the feasibility of growing crystals of protein in volumes as small as 1 nanoliter. Advances in the handling of very small volumes (i.e. through inkjet and other technologies) open the way towards fully automated systems. The rationale for these experiments is the desire to develop a system that speeds up the structure determination of proteins by crystallographic techniques, where most of the precious protein sample is wasted for the identification of the ideal crystallisation conditions. An additional potential benefit of crystallisation in very small volumes is the potential improvement of the crystal quality through reduced convection during crystal growth. Furthermore, in such small volumes even very highly supersaturated conditions can be stable for prolonged periods, allowing additional regions of phase-space to be prospected for elusive crystallisation conditions. A massive improvement in the efficiency of protein crystallogenesis will cause a paradigm shift in the biomolecular sciences and will have a major impact in product development in (for example) the pharmaceutical industry.

  4. Preparing Protein Samples

    NASA Technical Reports Server (NTRS)

    2000-01-01

    Cindy Barnes of University Space Research Association (USRA) at NASA's Marshall Space Flight Center pipettes a protein solution in preparation to grow crystals as part of NASA's structural biology program. Research on Earth helps scientists define conditions and specimens they will use in space experiments.

  5. The Protein Ensemble Database.

    PubMed

    Varadi, Mihaly; Tompa, Peter

    2015-01-01

    The scientific community's major conceptual notion of structural biology has recently shifted in emphasis from the classical structure-function paradigm due to the emergence of intrinsically disordered proteins (IDPs). As opposed to their folded cousins, these proteins are defined by the lack of a stable 3D fold and a high degree of inherent structural heterogeneity that is closely tied to their function. Due to their flexible nature, solution techniques such as small-angle X-ray scattering (SAXS), nuclear magnetic resonance (NMR) spectroscopy and fluorescence resonance energy transfer (FRET) are particularly well-suited for characterizing their biophysical properties. Computationally derived structural ensembles based on such experimental measurements provide models of the conformational sampling displayed by these proteins, and they may offer valuable insights into the functional consequences of inherent flexibility. The Protein Ensemble Database (http://pedb.vib.be) is the first openly accessible, manually curated online resource storing the ensemble models, protocols used during the calculation procedure, and underlying primary experimental data derived from SAXS and/or NMR measurements. By making this previously inaccessible data freely available to researchers, this novel resource is expected to promote the development of more advanced modelling methodologies, facilitate the design of standardized calculation protocols, and consequently lead to a better understanding of how function arises from the disordered state.

  6. Protein specific polymeric immunomicrospheres

    NASA Technical Reports Server (NTRS)

    Rembaum, Alan (Inventor); Yen, Shiao-Ping S. (Inventor); Dreyer, William J. (Inventor)

    1980-01-01

    Small, round, bio-compatible microspheres capable of covalently bonding proteins and having a uniform diameter below about 3500 A are prepared by substantially instantaneously initiating polymerization of an aqueous emulsion containing no more than 35% total monomer including an acrylic monomer substituted with a covalently bondable group such as hydroxyl, amino or carboxyl and a minor amount of a cross-linking agent.

  7. Tuber Storage Proteins

    PubMed Central

    SHEWRY, PETER R.

    2003-01-01

    A wide range of plants are grown for their edible tubers, but five species together account for almost 90 % of the total world production. These are potato (Solanum tuberosum), cassava (Manihot esculenta), sweet potato (Ipomoea batatus), yams (Dioscorea spp.) and taro (Colocasia, Cyrtosperma and Xanthosoma spp.). All of these, except cassava, contain groups of storage proteins, but these differ in the biological properties and evolutionary relationships. Thus, patatin from potato exhibits activity as an acylhydrolase and esterase, sporamin from sweet potato is an inhibitor of trypsin, and dioscorin from yam is a carbonic anhydrase. Both sporamin and dioscorin also exhibit antioxidant and radical scavenging activity. Taro differs from the other three crops in that it contains two major types of storage protein: a trypsin inhibitor related to sporamin and a mannose‐binding lectin. These characteristics indicate that tuber storage proteins have evolved independently in different species, which contrasts with the highly conserved families of storage proteins present in seeds. Furthermore, all exhibit biological activities which could contribute to resistance to pests, pathogens or abiotic stresses, indicating that they may have dual roles in the tubers. PMID:12730067

  8. Protein states and proteinquakes.

    PubMed Central

    Ansari, A; Berendzen, J; Bowne, S F; Frauenfelder, H; Iben, I E; Sauke, T B; Shyamsunder, E; Young, R D

    1985-01-01

    After photodissociation of carbon monoxide bound to myoglobin, the protein relaxes to the deoxy equilibrium structure in a quake-like motion. Investigation of the proteinquake and of related intramolecular equilibrium motions shows that states and motions have a hierarchical glass-like structure. PMID:3860839

  9. Protein Requirements during Aging.

    PubMed

    Courtney-Martin, Glenda; Ball, Ronald O; Pencharz, Paul B; Elango, Rajavel

    2016-01-01

    Protein recommendations for elderly, both men and women, are based on nitrogen balance studies. They are set at 0.66 and 0.8 g/kg/day as the estimated average requirement (EAR) and recommended dietary allowance (RDA), respectively, similar to young adults. This recommendation is based on single linear regression of available nitrogen balance data obtained at test protein intakes close to or below zero balance. Using the indicator amino acid oxidation (IAAO) method, we estimated the protein requirement in young adults and in both elderly men and women to be 0.9 and 1.2 g/kg/day as the EAR and RDA, respectively. This suggests that there is no difference in requirement on a gender basis or on a per kg body weight basis between younger and older adults. The requirement estimates however are ~40% higher than the current protein recommendations on a body weight basis. They are also 40% higher than our estimates in young men when calculated on the basis of fat free mass. Thus, current recommendations may need to be re-assessed. Potential rationale for this difference includes a decreased sensitivity to dietary amino acids and increased insulin resistance in the elderly compared with younger individuals. PMID:27529275

  10. Protein crystal growth

    NASA Technical Reports Server (NTRS)

    2001-01-01

    Atomic force microscopy uses laser technology to reveal a defect, a double-screw dislocation, on the surface of this crystal of canavalin, a major source of dietary protein for humans and domestic animals. When a crystal grows, attachment kinetics and transport kinetics are competing for control of the molecules. As a molecule gets close to the crystal surface, it has to attach properly for the crystal to be usable. NASA has funded investigators to look at those attachment kinetics from a theoretical standpoint and an experimental standpoint. Dr. Alex McPherson of the University of California, Irvine, is one of those investigators. He uses X-ray diffraction and atomic force microscopy in his laboratory to answer some of the many questions about how protein crystals grow. Atomic force microscopy provides a means of looking at how individual molecules are added to the surface of growing protein crystals. This helps McPherson understand the kinetics of protein crystal growth. McPherson asks, How fast do crystals grow? What are the forces involved? Investigators funded by NASA have clearly shown that such factors as the level of supersaturation and the rate of growth all affect the habit [characteristic arrangement of facets] of the crystal and the defects that occur in the crystal.

  11. Ribosome-inactivating proteins

    PubMed Central

    Walsh, Matthew J; Dodd, Jennifer E; Hautbergue, Guillaume M

    2013-01-01

    Ribosome-inactivating proteins (RIPs) were first isolated over a century ago and have been shown to be catalytic toxins that irreversibly inactivate protein synthesis. Elucidation of atomic structures and molecular mechanism has revealed these proteins to be a diverse group subdivided into two classes. RIPs have been shown to exhibit RNA N-glycosidase activity and depurinate the 28S rRNA of the eukaryotic 60S ribosomal subunit. In this review, we compare archetypal RIP family members with other potent toxins that abolish protein synthesis: the fungal ribotoxins which directly cleave the 28S rRNA and the newly discovered Burkholderia lethal factor 1 (BLF1). BLF1 presents additional challenges to the current classification system since, like the ribotoxins, it does not possess RNA N-glycosidase activity but does irreversibly inactivate ribosomes. We further discuss whether the RIP classification should be broadened to include toxins achieving irreversible ribosome inactivation with similar turnovers to RIPs, but through different enzymatic mechanisms. PMID:24071927

  12. Tuber storage proteins.

    PubMed

    Shewry, Peter R

    2003-06-01

    A wide range of plants are grown for their edible tubers, but five species together account for almost 90 % of the total world production. These are potato (Solanum tuberosum), cassava (Manihot esculenta), sweet potato (Ipomoea batatus), yams (Dioscorea spp.) and taro (Colocasia, Cyrtosperma and Xanthosoma spp.). All of these, except cassava, contain groups of storage proteins, but these differ in the biological properties and evolutionary relationships. Thus, patatin from potato exhibits activity as an acylhydrolase and esterase, sporamin from sweet potato is an inhibitor of trypsin, and dioscorin from yam is a carbonic anhydrase. Both sporamin and dioscorin also exhibit antioxidant and radical scavenging activity. Taro differs from the other three crops in that it contains two major types of storage protein: a trypsin inhibitor related to sporamin and a mannose-binding lectin. These characteristics indicate that tuber storage proteins have evolved independently in different species, which contrasts with the highly conserved families of storage proteins present in seeds. Furthermore, all exhibit biological activities which could contribute to resistance to pests, pathogens or abiotic stresses, indicating that they may have dual roles in the tubers.

  13. Cellulose binding domain proteins

    DOEpatents

    Shoseyov, O.; Shpiegl, I.; Goldstein, M.; Doi, R.

    1998-11-17

    A cellulose binding domain (CBD) having a high affinity for crystalline cellulose and chitin is disclosed, along with methods for the molecular cloning and recombinant production. Fusion products comprising the CBD and a second protein are likewise described. A wide range of applications are contemplated for both the CBD and the fusion products, including drug delivery, affinity separations, and diagnostic techniques. 16 figs.

  14. Protein Requirements during Aging

    PubMed Central

    Courtney-Martin, Glenda; Ball, Ronald O.; Pencharz, Paul B.; Elango, Rajavel

    2016-01-01

    Protein recommendations for elderly, both men and women, are based on nitrogen balance studies. They are set at 0.66 and 0.8 g/kg/day as the estimated average requirement (EAR) and recommended dietary allowance (RDA), respectively, similar to young adults. This recommendation is based on single linear regression of available nitrogen balance data obtained at test protein intakes close to or below zero balance. Using the indicator amino acid oxidation (IAAO) method, we estimated the protein requirement in young adults and in both elderly men and women to be 0.9 and 1.2 g/kg/day as the EAR and RDA, respectively. This suggests that there is no difference in requirement on a gender basis or on a per kg body weight basis between younger and older adults. The requirement estimates however are ~40% higher than the current protein recommendations on a body weight basis. They are also 40% higher than our estimates in young men when calculated on the basis of fat free mass. Thus, current recommendations may need to be re-assessed. Potential rationale for this difference includes a decreased sensitivity to dietary amino acids and increased insulin resistance in the elderly compared with younger individuals. PMID:27529275

  15. Dynamics of protein conformations

    NASA Astrophysics Data System (ADS)

    Stepanova, Maria

    2010-10-01

    A novel theoretical methodology is introduced to identify dynamic structural domains and analyze local flexibility in proteins. The methodology employs a multiscale approach combining identification of essential collective coordinates based on the covariance analysis of molecular dynamics trajectories, construction of the Mori projection operator with these essential coordinates, and analysis of the corresponding generalized Langevin equations [M.Stepanova, Phys.Rev.E 76(2007)051918]. Because the approach employs a rigorous theory, the outcomes are physically transparent: the dynamic domains are associated with regions of relative rigidity in the protein, whereas off-domain regions are relatively soft. This also allows scoring the flexibility in the macromolecule with atomic-level resolution [N.Blinov, M.Berjanskii, D.S.Wishart, and M.Stepanova, Biochemistry, 48(2009)1488]. The applications include the domain coarse-graining and characterization of conformational stability in protein G and prion proteins. The results are compared with published NMR experiments. Potential applications for structural biology, bioinformatics, and drug design are discussed.

  16. Cellulose binding domain proteins

    DOEpatents

    Shoseyov, Oded; Shpiegl, Itai; Goldstein, Marc; Doi, Roy

    1998-01-01

    A cellulose binding domain (CBD) having a high affinity for crystalline cellulose and chitin is disclosed, along with methods for the molecular cloning and recombinant production thereof. Fusion products comprising the CBD and a second protein are likewise described. A wide range of applications are contemplated for both the CBD and the fusion products, including drug delivery, affinity separations, and diagnostic techniques.

  17. [ALR, the multifunctional protein].

    PubMed

    Balogh, Tibor; Szarka, András

    2015-03-29

    ALR is a mystic protein. It has a so called "long" 22 kDa and a "short" 15 kDa forms. It has been described after partial hepatectomy and it has just been considered as a key protein of liver regeneration. At the beginning of the 21st century it has been revealed that the "long" form is localized in the mitochondrial intermembrane space and it is an element of the mitochondrial protein import and disulphide relay system. Several proteins of the substrates of the mitochondrial disulphide relay system are necessary for the proper function of the mitochondria, thus any mutation of the ALR gene leads to mitochondrial diseases. The "short" form of ALR functions as a secreted extracellular growth factor and it promotes the protection, regeneration and proliferation of hepatocytes. The results gained on the recently generated conditional ALR mutant mice suggest that ALR can play an important role in the pathogenesis of alcoholic and non-alcoholic steatosis. Since the serum level of ALR is modified in several liver diseases it can be a promising marker molecule in laboratory diagnostics. PMID:25796277

  18. Chaos in protein dynamics.

    PubMed

    Braxenthaler, M; Unger, R; Auerbach, D; Given, J A; Moult, J

    1997-12-01

    MD simulations, currently the most detailed description of the dynamic evolution of proteins, are based on the repeated solution of a set of differential equations implementing Newton's second law. Many such systems are known to exhibit chaotic behavior, i.e., very small changes in initial conditions are amplified exponentially and lead to vastly different, inherently unpredictable behavior. We have investigated the response of a protein fragment in an explicit solvent environment to very small perturbations of the atomic positions (10(-3)-10(-9) A). Independent of the starting conformation (native-like, compact, extended), perturbed dynamics trajectories deviated rapidly, leading to conformations that differ by approximately 1 A RMSD within 1-2 ps. Furthermore, introducing the perturbation more than 1-2 ps before a significant conformational transition leads to a loss of the transition in the perturbed trajectories. We present evidence that the observed chaotic behavior reflects physical properties of the system rather than numerical instabilities of the calculation and discuss the implications for models of protein folding and the use of MD as a tool to analyze protein folding pathways.

  19. Protein stability in ice.

    PubMed

    Strambini, Giovanni B; Gonnelli, Margherita

    2007-03-15

    This study presents an experimental approach, based on the change of Trp fluorescence between native and denatured states of proteins, which permits to monitor unfolding equilibria and the thermodynamic stability (DeltaG degrees ) of these macromolecules in frozen aqueous solutions. The results obtained by guanidinium chloride denaturation of the azurin mutant C112S from Pseudomonas aeruginosa, in the temperature range from -8 to -16 degrees C, demonstrate that the stability of the native fold may be significantly perturbed in ice depending mainly on the size of the liquid water pool (V(L)) in equilibrium with the solid phase. The data establish a threshold, around V(L)=1.5%, below which in ice DeltaG degrees decreases progressively relative to liquid state, up to 3 kcal/mole for V(L)=0.285%. The sharp dependence of DeltaG degrees on V(L) is consistent with a mechanism based on adsorption of the protein to the ice surface. The reduction in DeltaG degrees is accompanied by a corresponding decrease in m-value indicating that protein-ice interactions increase the solvent accessible surface area of the native fold or reduce that of the denatured state, or both. The method opens the possibility for examining in a more quantitative fashion the influence of various experimental conditions on the ice perturbation and in particular to test the effectiveness of numerous additives used in formulations to preserve labile pharmaco proteins.

  20. Weakly Stable Regions and Protein-Protein Interactions in Beta-Barrel Membrane Proteins

    PubMed Central

    Naveed, Hammad; Liang, Jie

    2014-01-01

    We briefly discuss recent progress in computational characterization of the sequence and structural properties of β-barrel membrane properties. We discuss the emerging concept of weakly stable regions in β-barrel membrane proteins, computational methods to identify these regions and mechanisms adopted by β-barrel membrane proteins in nature to stabilize them. We further discuss computational methods to identify protein-protein interactions in β-barrel membrane proteins and recent experimental studies that aim at altering the biophysical properties including oligomerization state and stability of β-barrel membrane proteins based on the emerging organization principles of these proteins from recent computational studies. PMID:23713778

  1. Protein farnesyltransferase inhibitors.

    PubMed

    Ayral-Kaloustian, Semiramis; Salaski, Edward J

    2002-05-01

    Specific mutations in the ras gene impair the guanosine triphophatase (GTPase) activity of Ras proteins, which play a fundamental role in the signaling cascade, leading to uninterrupted growth signals and to the transformation of normal cells into malignant phenotypes. It has been shown that normal cells transfected with mutant ras gene become cancerous and that unfarnesylated, cytosolic mutant Ras protein does not anchor onto cell membranes and cannot induce this transformation. Posttranslational modification and plasma membrane association of mutant Ras is necessary for this transforming activity. Since its identification, the enzyme protein farnesyltransferase (FTase) that catalyzes the first and essential step of the three Ras-processing steps has emerged as the most promising target for therapeutic intervention. FTase has been implicated as a potential target in inhibiting the prenylation of a variety of proteins, thus in controlling varied disease states (e.g. cancer, neurofibromatosis, restenosis, viral hepatitis, bone resorption, parasitic infections, corneal inflammations, and diabetes) associated with prenyl modifications of Ras and other proteins. Furthermore, it has been suggested that FTase inhibitors indirectly help in inhibiting tumors via suppression of angiogenesis and induction of apoptosis. Major milestones have been achieved with small-molecule FTase inhibitors that show efficacy without toxicity in vitro, as well as in mouse models bearing ras-dependent tumors. With the determination of the crystal structure of mammalian FTase, existent leads have been fine-tuned and new potent molecules of diverse structural classes have been designed. A few of these molecules are currently in the clinic, with at least three drug candidates in Phase II studies and one in Phase III. This article will review the progress that has been reported with FTase inhibitors in drug discovery and in the clinic. PMID:12733981

  2. Transglutaminase-mediated oligomerization promotes osteoblast adhesive properties of osteopontin and bone sialoprotein

    PubMed Central

    Forsprecher, Jennifer; Wang, Zhemeng; Goldberg, Harvey A

    2011-01-01

    Tissue transglutaminase (TG2) is a widely distributed, protein-crosslinking enzyme having a prominent role in cell adhesion as a β1 integrin co-receptor for fibronectin. In bone and teeth, its substrates include the matricellular proteins osteopontin (OPN) and bone sialoprotein (BSP). The aim of this study was to examine effects of TG2-mediated crosslinking and oligomerization of OPN and BSP on osteoblast cell adhesion. We show that surfaces coated with oligomerized OPN and BSP promote MC3T3-E1/C4 osteoblastic cell adhesion significantly better than surfaces coated with the monomeric form of the proteins. Both OPN and BSP oligomer-adherent cells showed more cytoplasmic extensions than those cells grown on the monomer-coated surfaces indicative of increased cell connectivity. Our study suggests a role for TG2 in promoting the cell adhesion function of two matricellular substrate proteins prominent in bone, tooth cementum and certain tumors. PMID:20864802

  3. Bayesian Estimator of Protein-Protein Association Probabilities

    2008-05-28

    The Bayesian Estimator of Protein-Protein Association Probabilities (BEPro3) is a software tool for estimating probabilities of protein-protein association between bait and prey protein pairs using data from multiple-bait, multiple-replicate, protein LC-MS/MS affinity isolation experiments. BEPro3 is public domain software, has been tested on Windows XP and version 10.4 or newer of the Mac OS 10.4, and is freely available. A user guide, example dataset with analysis and additional documentation are included with the BEPro3 download.

  4. Direct Probing of Protein-Protein Interactions

    SciTech Connect

    Noy, A; Sulchek, T A; Friddle, R W

    2005-03-10

    This project aimed to establish feasibility of using experimental techniques based on direct measurements of interaction forces on the single molecule scale to characterize equilibrium interaction potentials between individual biological molecules. Such capability will impact several research areas, ranging from rapid interaction screening capabilities to providing verifiable inputs for computational models. It should be one of the enabling technologies for modern proteomics research. This study used a combination of Monte-Carlo simulations, theoretical considerations, and direct experimental measurements to investigate two model systems that represented typical experimental situations: force-induced melting of DNA rigidly attached to the tip, and force-induced unbinding of a protein-antibody pair connected to flexible tethers. Our results establish that for both systems researchers can use force spectroscopy measurements to extract reliable information about equilibrium interaction potentials. However, the approaches necessary to extract these potentials in each case--Jarzynski reconstruction and Dynamic Force Spectroscopy--are very different. We also show how the thermodynamics and kinetics of unbinding process dictates the choice between in each case.

  5. Protein-protein interaction databases: keeping up with growing interactomes

    PubMed Central

    2009-01-01

    Over the past few years, the number of known protein-protein interactions has increased substantially. To make this information more readily available, a number of publicly available databases have set out to collect and store protein-protein interaction data. Protein-protein interactions have been retrieved from six major databases, integrated and the results compared. The six databases (the Biological General Repository for Interaction Datasets [BioGRID], the Molecular INTeraction database [MINT], the Biomolecular Interaction Network Database [BIND], the Database of Interacting Proteins [DIP], the IntAct molecular interaction database [IntAct] and the Human Protein Reference Database [HPRD]) differ in scope and content; integration of all datasets is non-trivial owing to differences in data annotation. With respect to human protein-protein interaction data, HPRD seems to be the most comprehensive. To obtain a complete dataset, however, interactions from all six databases have to be combined. To overcome this limitation, meta-databases such as the Agile Protein Interaction Database (APID) offer access to integrated protein-protein interaction datasets, although these also currently have certain restrictions. PMID:19403463

  6. Identifying the hub proteins from complicated membrane protein network systems.

    PubMed

    Shen, Yi-Zhen; Ding, Yong-Sheng; Gu, Quan; Chou, Kuo-Chen

    2010-05-01

    The so-called "hub proteins" are those proteins in a protein-protein interaction network system that have remarkably higher interaction relations (or degrees) than the others. Therefore, the information of hub proteins can provide very useful insights for selecting or prioritizing targets during drug development. In this paper, by combining the multi-agent-based method with the graphical spectrum analysis and immune-genetic algorithm, a novel simulator for identifying the hub proteins from membrane protein interaction networks is proposed. As a demonstration of using the simulator, two hub membrane proteins, YPL227C and YIL147C, were identified from a complicated network system consisting of 1500 membrane proteins. Meanwhile, along with the two identified hub proteins, their molecular functions, biological processes, and cellular components were also revealed. It is anticipated that the hub-protein-simulator may become a very useful tool for system biology and drug development, particularly in deciphering unknown protein functions, determining protein complexes, and in identifying the key targets from a complicated disease system. PMID:20507268

  7. Molecular simulations of lipid-mediated protein-protein interactions.

    PubMed

    de Meyer, Frédérick Jean-Marie; Venturoli, Maddalena; Smit, Berend

    2008-08-01

    Recent experimental results revealed that lipid-mediated interactions due to hydrophobic forces may be important in determining the protein topology after insertion in the membrane, in regulating the protein activity, in protein aggregation and in signal transduction. To gain insight into the lipid-mediated interactions between two intrinsic membrane proteins, we developed a mesoscopic model of a lipid bilayer with embedded proteins, which we studied with dissipative particle dynamics. Our calculations of the potential of mean force between transmembrane proteins show that hydrophobic forces drive long-range protein-protein interactions and that the nature of these interactions depends on the length of the protein hydrophobic segment, on the three-dimensional structure of the protein and on the properties of the lipid bilayer. To understand the nature of the computed potentials of mean force, the concept of hydrophilic shielding is introduced. The observed protein interactions are interpreted as resulting from the dynamic reorganization of the system to maintain an optimal hydrophilic shielding of the protein and lipid hydrophobic parts, within the constraint of the flexibility of the components. Our results could lead to a better understanding of several membrane processes in which protein interactions are involved. PMID:18487292

  8. Redox control of protein degradation

    PubMed Central

    Pajares, Marta; Jiménez-Moreno, Natalia; Dias, Irundika H.K.; Debelec, Bilge; Vucetic, Milica; Fladmark, Kari E.; Basaga, Huveyda; Ribaric, Samo; Milisav, Irina; Cuadrado, Antonio

    2015-01-01

    Intracellular proteolysis is critical to maintain timely degradation of altered proteins including oxidized proteins. This review attempts to summarize the most relevant findings about oxidant protein modification, as well as the impact of reactive oxygen species on the proteolytic systems that regulate cell response to an oxidant environment: the ubiquitin-proteasome system (UPS), autophagy and the unfolded protein response (UPR). In the presence of an oxidant environment, these systems are critical to ensure proteostasis and cell survival. An example of altered degradation of oxidized proteins in pathology is provided for neurodegenerative diseases. Future work will determine if protein oxidation is a valid target to combat proteinopathies. PMID:26381917

  9. Hydrogels Constructed from Engineered Proteins.

    PubMed

    Li, Hongbin; Kong, Na; Laver, Bryce; Liu, Junqiu

    2016-02-24

    Due to their various potential biomedical applications, hydrogels based on engineered proteins have attracted considerable interest. Benefitting from significant progress in recombinant DNA technology and protein engineering/design techniques, the field of protein hydrogels has made amazing progress. The latest progress of hydrogels constructed from engineered recombinant proteins are presented, mainly focused on biorecognition-driven physical hydrogels as well as chemically crosslinked hydrogels. The various bio-recognition based physical crosslinking strategies are discussed, as well as chemical crosslinking chemistries used to engineer protein hydrogels, and protein hydrogels' various biomedical applications. The future perspectives of this fast evolving field of biomaterials are also discussed. PMID:26707834

  10. Hydrogels Constructed from Engineered Proteins.

    PubMed

    Li, Hongbin; Kong, Na; Laver, Bryce; Liu, Junqiu

    2016-02-24

    Due to their various potential biomedical applications, hydrogels based on engineered proteins have attracted considerable interest. Benefitting from significant progress in recombinant DNA technology and protein engineering/design techniques, the field of protein hydrogels has made amazing progress. The latest progress of hydrogels constructed from engineered recombinant proteins are presented, mainly focused on biorecognition-driven physical hydrogels as well as chemically crosslinked hydrogels. The various bio-recognition based physical crosslinking strategies are discussed, as well as chemical crosslinking chemistries used to engineer protein hydrogels, and protein hydrogels' various biomedical applications. The future perspectives of this fast evolving field of biomaterials are also discussed.

  11. Motif-Driven Design of Protein-Protein Interfaces.

    PubMed

    Silva, Daniel-Adriano; Correia, Bruno E; Procko, Erik

    2016-01-01

    Protein-protein interfaces regulate many critical processes for cellular function. The ability to accurately control and regulate these molecular interactions is of major interest for biomedical and synthetic biology applications, as well as to address fundamental biological questions. In recent years, computational protein design has emerged as a tool for designing novel protein-protein interactions with functional relevance. Although attractive, these computational tools carry a steep learning curve. In order to make some of these methods more accessible, we present detailed descriptions and examples of ROSETTA computational protocols for the design of functional protein binders using seeded protein interface design. In these protocols, a motif of known structure that interacts with the target site is grafted into a scaffold protein, followed by design of the surrounding interaction surface. PMID:27094298

  12. How do oncoprotein mutations rewire protein-protein interaction networks?

    PubMed

    Bowler, Emily H; Wang, Zhenghe; Ewing, Rob M

    2015-01-01

    The acquisition of mutations that activate oncogenes or inactivate tumor suppressors is a primary feature of most cancers. Mutations that directly alter protein sequence and structure drive the development of tumors through aberrant expression and modification of proteins, in many cases directly impacting components of signal transduction pathways and cellular architecture. Cancer-associated mutations may have direct or indirect effects on proteins and their interactions and while the effects of mutations on signaling pathways have been widely studied, how mutations alter underlying protein-protein interaction networks is much less well understood. Systematic mapping of oncoprotein protein interactions using proteomics techniques as well as computational network analyses is revealing how oncoprotein mutations perturb protein-protein interaction networks and drive the cancer phenotype. PMID:26325016

  13. Functionalizing Microporous Membranes for Protein Purification and Protein Digestion

    NASA Astrophysics Data System (ADS)

    Dong, Jinlan; Bruening, Merlin L.

    2015-07-01

    This review examines advances in the functionalization of microporous membranes for protein purification and the development of protease-containing membranes for controlled protein digestion prior to mass spectrometry analysis. Recent studies confirm that membranes are superior to bead-based columns for rapid protein capture, presumably because convective mass transport in membrane pores rapidly brings proteins to binding sites. Modification of porous membranes with functional polymeric films or TiO2 nanoparticles yields materials that selectively capture species ranging from phosphopeptides to His-tagged proteins, and protein-binding capacities often exceed those of commercial beads. Thin membranes also provide a convenient framework for creating enzyme-containing reactors that afford control over residence times. With millisecond residence times, reactors with immobilized proteases limit protein digestion to increase sequence coverage in mass spectrometry analysis and facilitate elucidation of protein structures. This review emphasizes the advantages of membrane-based techniques and concludes with some challenges for their practical application.

  14. Electron flow through proteins

    NASA Astrophysics Data System (ADS)

    Gray, Harry B.; Winkler, Jay R.

    2009-11-01

    Electron transfers in photosynthesis and respiration commonly occur between metal-containing cofactors that are separated by large molecular distances. Employing laser flash-quench triggering methods, we have shown that 20-Å, coupling-limited Fe II-Ru III and Cu I-Ru III electron tunneling in Ru-modified cytochromes and blue copper proteins can occur on the microsecond timescale both in solutions and crystals. Redox equivalents can be transferred even longer distances by multistep tunneling, often called hopping, through intervening amino acid side chains. Our work has established that 20-Å hole hopping through an intervening tryptophan is two orders of magnitude faster than single-step electron tunneling in a Re-modified blue copper protein.

  15. A magnetic protein biocompass

    NASA Astrophysics Data System (ADS)

    Qin, Siying; Yin, Hang; Yang, Celi; Dou, Yunfeng; Liu, Zhongmin; Zhang, Peng; Yu, He; Huang, Yulong; Feng, Jing; Hao, Junfeng; Hao, Jia; Deng, Lizong; Yan, Xiyun; Dong, Xiaoli; Zhao, Zhongxian; Jiang, Taijiao; Wang, Hong-Wei; Luo, Shu-Jin; Xie, Can

    2016-02-01

    The notion that animals can detect the Earth’s magnetic field was once ridiculed, but is now well established. Yet the biological nature of such magnetosensing phenomenon remains unknown. Here, we report a putative magnetic receptor (Drosophila CG8198, here named MagR) and a multimeric magnetosensing rod-like protein complex, identified by theoretical postulation and genome-wide screening, and validated with cellular, biochemical, structural and biophysical methods. The magnetosensing complex consists of the identified putative magnetoreceptor and known magnetoreception-related photoreceptor cryptochromes (Cry), has the attributes of both Cry- and iron-based systems, and exhibits spontaneous alignment in magnetic fields, including that of the Earth. Such a protein complex may form the basis of magnetoreception in animals, and may lead to applications across multiple fields.

  16. Protein Structure Databases.

    PubMed

    Laskowski, Roman A

    2016-01-01

    Web-based protein structure databases come in a wide variety of types and levels of information content. Those having the most general interest are the various atlases that describe each experimentally determined protein structure and provide useful links, analyses, and schematic diagrams relating to its 3D structure and biological function. Also of great interest are the databases that classify 3D structures by their folds as these can reveal evolutionary relationships which may be hard to detect from sequence comparison alone. Related to these are the numerous servers that compare folds-particularly useful for newly solved structures, and especially those of unknown function. Beyond these are a vast number of databases for the more specialized user, dealing with specific families, diseases, structural features, and so on. PMID:27115626

  17. Cow's Milk Protein Allergy.

    PubMed

    Mousan, Grace; Kamat, Deepak

    2016-10-01

    Cow's milk protein allergy (CMPA) is a common condition encountered in children with incidence estimated as 2% to 7.5% in the first year of life. Formula and breast-fed babies can present with symptoms of CMPA. It is important to accurately diagnose CMPA to avoid the consequences of either under- or overdiagnosis. CMPA is classically categorized into immunoglobulin E (IgE)- or non-IgE-mediated reaction that vary in clinical manifestations, diagnostic evaluation, and prognosis. The most commonly involved systems in patients with CMPA are gastrointestinal, skin, and respiratory. Evaluation of CMPA starts with good data gathering followed by testing if indicated. Treatment is simply by avoidance of cow's milk protein (CMP) in the child's or mother's diet, if exclusively breast-feeding. This article reviews the definition, epidemiology, risk factors, pathogenesis, clinical presentation, evaluation, management, and prognosis of CMPA and provides an overview of different options for formulas and their indication in the treatment of CMPA.

  18. Purine inhibitors of protein kinases, G proteins and polymerases

    DOEpatents

    Gray, Nathanael S.; Schultz, Peter; Kim, Sung-Hou; Meijer, Laurent

    2004-10-12

    The present invention relates to 2-N-substituted 6-(4-methoxybenzylamino)-9-isopropylpurines that inhibit, inter alia, protein kinases, G-proteins and polymerases. In addition, the present invention relates to methods of using such 2-N-substituted 6-(4-methoxybenzylamino)-9-isopropylpurines to inhibit protein kinases, G-proteins, polymerases and other cellular processes and to treat cellular proliferative diseases.

  19. Protein-protein fusion catalyzed by sortase A.

    PubMed

    Levary, David A; Parthasarathy, Ranganath; Boder, Eric T; Ackerman, Margaret E

    2011-04-06

    Chimeric proteins boast widespread use in areas ranging from cell biology to drug delivery. Post-translational protein fusion using the bacterial transpeptidase sortase A provides an attractive alternative when traditional gene fusion fails. We describe use of this enzyme for in vitro protein ligation and report the successful fusion of 10 pairs of protein domains with preserved functionality--demonstrating the robust and facile nature of this reaction.

  20. Protein-Protein Fusion Catalyzed by Sortase A

    PubMed Central

    Levary, David A.; Parthasarathy, Ranganath; Boder, Eric T.; Ackerman, Margaret E.

    2011-01-01

    Chimeric proteins boast widespread use in areas ranging from cell biology to drug delivery. Post-translational protein fusion using the bacterial transpeptidase sortase A provides an attractive alternative when traditional gene fusion fails. We describe use of this enzyme for in vitro protein ligation and report the successful fusion of 10 pairs of protein domains with preserved functionality — demonstrating the robust and facile nature of this reaction. PMID:21494692

  1. Predicting disease-related proteins based on clique backbone in protein-protein interaction network.

    PubMed

    Yang, Lei; Zhao, Xudong; Tang, Xianglong

    2014-01-01

    Network biology integrates different kinds of data, including physical or functional networks and disease gene sets, to interpret human disease. A clique (maximal complete subgraph) in a protein-protein interaction network is a topological module and possesses inherently biological significance. A disease-related clique possibly associates with complex diseases. Fully identifying disease components in a clique is conductive to uncovering disease mechanisms. This paper proposes an approach of predicting disease proteins based on cliques in a protein-protein interaction network. To tolerate false positive and negative interactions in protein networks, extending cliques and scoring predicted disease proteins with gene ontology terms are introduced to the clique-based method. Precisions of predicted disease proteins are verified by disease phenotypes and steadily keep to more than 95%. The predicted disease proteins associated with cliques can partly complement mapping between genotype and phenotype, and provide clues for understanding the pathogenesis of serious diseases.

  2. Path to protein crystallization

    SciTech Connect

    2010-01-01

    Growth of two-dimensional S-layer crystals on supported lipid bilayers observed in solution using in situ atomic force microscopy. This movie shows proteins sticking onto the supported lipid bilayer, forming a mobile phase that condenses into amorphous clusters, and undergoing a phase transition to crystalline clusters composed of 2 to 15 tetramers. These initial clusters then enter a growth phase in which new tetramers form exclusively at unoccupied lattice sites along the cluster edges.

  3. Microdosing of protein drugs.

    PubMed

    Rowland, M

    2016-02-01

    Poor pharmacokinetics (PK) can seriously limit clinical utility. Knowing early whether a new compound is likely to have the desired PK profile at therapeutic doses is therefore important. One approach, microdosing, has shown high success with small molecular weight compounds, despite early skepticism. Vlaming et al. report the first, and successful, clinical application of a microdose of a humanized recombinant protein. But what is the likely success for this class of drugs more generally?

  4. Myosin V motor proteins

    PubMed Central

    Vale, Ronald D.

    2003-01-01

    Mammalian myosin V motors transport cargo processively along actin filaments. Recent biophysical and structural studies have led to a detailed understanding of the mechanism of myosin V, making it perhaps the best understood cytoskeletal motor. In addition to describing the mechanism, this review will illustrate how “dynamic” single molecule measurements can synergize with “static” protein structural studies to produce amazingly clear information on the workings of a nanometer-scale machine. PMID:14610051

  5. Protein Crystal Isocitrate Lyase

    NASA Technical Reports Server (NTRS)

    1998-01-01

    The comparison of protein crystal, Isocitrate Lyase earth-grown (left) and space-grown (right). This is a target enzyme for fungicides. A better understanding of this enzyme should lead to the discovery of more potent fungicides to treat serious crop diseases such as rice blast; it regulates the flow of metabolic intermediates required for cell growth. Principal Investigator is Larry DeLucas.

  6. HRTEM in protein crystallography

    NASA Astrophysics Data System (ADS)

    Dyson, P. W.; Spargo, A. E. C.; Tulloch, P. A.; Johnson, A. W. S.

    Electron microscopy/diffraction (ED/D) using spot-scan and low-dose imaging has been successfully applied to investigate microcrystals of an alpha-helical coiled-coil protein extracted from ootheca of the praying mantis. Fourier transforms of the images show resolution out to 4 A and can be used to phase the corresponding ED data which shows reflections out to 2 A.

  7. Bone morphogenetic protein

    SciTech Connect

    Xiao Yongtao; Xiang Lixin; Shao Jianzhong

    2007-10-26

    Bone morphogenetic proteins (BMPs) are multi-functional growth factors belonging to the transforming growth factor-beta superfamily. It has been demonstrated that BMPs had been involved in the regulation of cell proliferation, survival, differentiation and apoptosis. However, their hallmark ability is that play a pivotal role in inducing bone, cartilage, ligament, and tendon formation at both heterotopic and orthotopic sites. In this review, we mainly concentrate on BMP structure, function, molecular signaling and potential medical application.

  8. Understanding Protein Non-Folding

    PubMed Central

    Uversky, Vladimir N.; Dunker, A. Keith

    2010-01-01

    This review describes the family of intrinsically disordered proteins, members of which fail to form rigid 3-D structures under physiological conditions, either along their entire lengths or only in localized regions. Instead, these intriguing proteins/regions exist as dynamic ensembles within which atom positions and backbone Ramachandran angles exhibit extreme temporal fluctuations without specific equilibrium values. Many of these intrinsically disordered proteins are known to carry out important biological functions which, in fact, depend on the absence of specific 3-D structure. The existence of such proteins does not fit the prevailing structure-function paradigm, which states that unique 3-D structure is a prerequisite to function. Thus, the protein structure-function paradigm has to be expanded to include intrinsically disordered proteins and alternative relationships among protein sequence, structure, and function. This shift in the paradigm represents a major breakthrough for biochemistry, biophysics and molecular biology, as it opens new levels of understanding with regard to the complex life of proteins. This review will try to answer the following questions: How were intrinsically disordered proteins discovered? Why don't these proteins fold? What is so special about intrinsic disorder? What are the functional advantages of disordered proteins/regions? What is the functional repertoire of these proteins? What are the relationships between intrinsically disordered proteins and human diseases? PMID:20117254

  9. Food allergy to proteins.

    PubMed

    Nowak-Wegrzyn, Anna

    2007-01-01

    Food allergy is defined as an immune system-mediated adverse reaction to food proteins. Class 1 food allergens are represented by peanut, egg white, and cow's milk; they are heat- and acid-stable glycoproteins that induce allergic sensitization via gastrointestinal tract and cause systemic reactions. Class 2 food allergens are homologous to proteins in birch tree pollen and class 2 food allergy develops as a consequence of respiratory sensitization to the cross-reactive pollen. Class 2 food allergens are very heat-labile and tend to induce reactions limited to oral allergy symptoms. In contrast, plant nonspecific lipid transfer proteins are resistant to heating and tend to induce systemic reactions. Analysis of IgE-binding epitopes with SPOT membranes revealed that cow's milk-, egg- and peanut-allergic subjects without IgE antibodies against certain sequential epitopes of the major allergens were more likely to achieve tolerance than subjects whose IgE antibodies were directed against those epitopes. Subsequently, peptide microarray showed a correlation between reaction severity and the intensity of IgE binding and the number of epitopes recognized of patients' immune responses against peanut allergens. Taken together, these data suggest that the epitope recognition pattern and intensity of IgE binding are important determinants of severity and duration of food allergy.

  10. Hydrolyzed Proteins in Allergy.

    PubMed

    Salvatore, Silvia; Vandenplas, Yvan

    2016-01-01

    Hydrolyzed proteins are used worldwide in the therapeutic management of infants with allergic manifestations and have long been proposed as a dietetic measure to prevent allergy in at risk infants. The degree and method of hydrolysis, protein source and non-nitrogen components characterize different hydrolyzed formulas (HFs) and may determine clinical efficacy, tolerance and nutritional effects. Cow's milk (CM)-based HFs are classified as extensively (eHF) or partially HF (pHF) based on the percentage of small peptides. One whey pHF has been shown to reduce atopic dermatitis in high-risk infants who are not exclusively breastfed. More studies are needed to determine the benefit of these formulas in the prevention of CM allergy (CMA) and in the general population. eHFs represent up to now the treatment of choice for most infants with CMA. However, new developments, such as an extensively hydrolyzed rice protein-based formula, could become alternative options if safety and nutritional and therapeutic efficacy are confirmed as this type of formula is less expensive. In some countries, an extensive soy hydrolysate is available. PMID:27336625

  11. Proteins Among the Polysaccharides

    PubMed Central

    Wen, Fushi; Curlango-Rivera, Gilberto

    2007-01-01

    Charles Darwin recognized the power of the root cap as a model for plant signalling and behavior, and used it to explore the ways plants sense and respond to diverse stimuli. Over ensuing decades, various groups have reported tantalizing clues regarding the role of a complex extracellular matrix that ensheaths the tip region housing the apical and root cap meristems. In the course of characterizing root tip resistance to infection and injury and the role border cells play in this phenomenon, we confirmed and extended early- and mid-20th century studies reporting enzyme activities secreted from the root cap. Multidimensional protein analysis revealed, in fact, that >100 proteins are actively synthesized and secreted from the root cap and border cells. This ‘root cap secretome’ appears to be a critical component of root tip resistance to infection. We have developed a microscopic assay to quantify the protein-based extracellular response to dynamic changes in environmental conditions including hydroponic culture, and present the results here. This tool provides a simple, direct measure that can be used to explore the ways border cells may function in the manner of white blood cells to trap, immobilize and neutralize threats to the growing root tip. PMID:19704617

  12. Process for protein PEGylation.

    PubMed

    Pfister, David; Morbidelli, Massimo

    2014-04-28

    PEGylation is a versatile drug delivery technique that presents a particularly wide range of conjugation chemistry and polymer structure. The conjugated protein can be tuned to specifically meet the needs of the desired application. In the area of drug delivery this typically means to increase the persistency in the human body without affecting the activity profile of the original protein. On the other hand, because of the high costs associated with the production of therapeutic proteins, subsequent operations imposed by PEGylation must be optimized to minimize the costs inherent to the additional steps. The closest attention has to be given to the PEGylation reaction engineering and to the subsequent purification processes. This review article focuses on these two aspects and critically reviews the current state of the art with a clear focus on the development of industrial scale processes which can meet the market requirements in terms of quality and costs. The possibility of using continuous processes, with integration between the reaction and the separation steps is also illustrated.

  13. Infrared Protein Crystallography

    SciTech Connect

    J Sage; Y Zhang; J McGeehan; R Ravelli; M Weik; J van Thor

    2011-12-31

    We consider the application of infrared spectroscopy to protein crystals, with particular emphasis on exploiting molecular orientation through polarization measurements on oriented single crystals. Infrared microscopes enable transmission measurements on individual crystals using either thermal or nonthermal sources, and can accommodate flow cells, used to measure spectral changes induced by exposure to soluble ligands, and cryostreams, used for measurements of flash-cooled crystals. Comparison of unpolarized infrared measurements on crystals and solutions probes the effects of crystallization and can enhance the value of the structural models refined from X-ray diffraction data by establishing solution conditions under which they are most relevant. Results on several proteins are consistent with similar equilibrium conformational distributions in crystal and solutions. However, the rates of conformational change are often perturbed. Infrared measurements also detect products generated by X-ray exposure, including CO{sub 2}. Crystals with favorable symmetry exhibit infrared dichroism that enhances the synergy with X-ray crystallography. Polarized infrared measurements on crystals can distinguish spectral contributions from chemically similar sites, identify hydrogen bonding partners, and, in opportune situations, determine three-dimensional orientations of molecular groups. This article is part of a Special Issue entitled: Protein Structure and Function in the Crystalline State.

  14. A polymetamorphic protein

    PubMed Central

    Stewart, Katie L; Dodds, Eric D; Wysocki, Vicki H; Cordes, Matthew H J

    2013-01-01

    Arc repressor is a homodimeric protein with a ribbon-helix–helix fold. A single polar-to-hydrophobic substitution (N11L) at a solvent-exposed position leads to population of an alternate dimeric fold in which 310 helices replace a β-sheet. Here we find that the variant Q9V/N11L/R13V (S-VLV), with two additional polar-to-hydrophobic surface mutations in the same β-sheet, forms a highly stable, reversibly folded octamer with approximately half the✠α-helical content of wild-type Arc. At low protein concentration and low ionic strength, S-VLV also populates both dimeric topologies previously observed for N11L, as judged by NMR chemical shift comparisons. Thus, accumulation of simple hydrophobic mutations in Arc progressively reduces fold specificity, leading first to a sequence with two folds and then to a manifold bridge sequence with at least three different topologies. Residues 9–14 of S-VLV form a highly hydrophobic stretch that is predicted to be amyloidogenic, but we do not observe aggregates of higher order than octamer. Increases in sequence hydrophobicity can promote amyloid aggregation but also exert broader and more complex effects on fold specificity. Altered native folds, changes in fold coupled to oligomerization, toxic pre-amyloid oligomers, and amyloid fibrils may represent a near continuum of accessible alternatives in protein structure space. PMID:23471712

  15. Papillomavirus E6 proteins

    SciTech Connect

    Howie, Heather L.; Katzenellenbogen, Rachel A.; Galloway, Denise A.

    2009-02-20

    The papillomaviruses are small DNA viruses that encode approximately eight genes, and require the host cell DNA replication machinery for their viral DNA replication. Thus papillomaviruses have evolved strategies to induce host cell DNA synthesis balanced with strategies to protect the cell from unscheduled replication. While the papillomavirus E1 and E2 genes are directly involved in viral replication by binding to and unwinding the origin of replication, the E6 and E7 proteins have auxillary functions that promote proliferation. As a consequence of disrupting the normal checkpoints that regulate cell cycle entry and progression, the E6 and E7 proteins play a key role in the oncogenic properties of human papillomaviruses with a high risk of causing anogenital cancers (HR HPVs). As a consequence, E6 and E7 of HR HPVs are invariably expressed in cervical cancers. This article will focus on the E6 protein and its numerous activities including inactivating p53, blocking apoptosis, activating telomerase, disrupting cell adhesion, polarity and epithelial differentiation, altering transcription and reducing immune recognition.

  16. Protein-protein interactions in the synaptonemal complex.

    PubMed Central

    Tarsounas, M; Pearlman, R E; Gasser, P J; Park, M S; Moens, P B

    1997-01-01

    In mammalian systems, an approximately M(r) 30,000 Cor1 protein has been identified as a major component of the meiotic prophase chromosome cores, and a M(r) 125,000 Syn1 protein is present between homologue cores where they are synapsed and form the synaptonemal complex (SC). Immunolocalization of these proteins during meiosis suggests possible homo- and heterotypic interactions between the two as well as possible interactions with yet unrecognized proteins. We used the two-hybrid system in the yeast Saccharomyces cerevisiae to detect possible protein-protein associations. Segments of hamsters Cor1 and Syn1 proteins were tested in various combinations for homo- and heterotypic interactions. In the cause of Cor1, homotypic interactions involve regions capable of coiled-coil formation, observation confirmed by in vitro affinity coprecipitation experiments. The two-hybrid assay detects no interaction of Cor1 protein with central and C-terminal fragments of Syn1 protein and no homotypic interactions involving these fragments of Syn1. Hamster Cor1 and Syn1 proteins both associate with the human ubiquitin-conjugation enzyme Hsubc9 as well as with the hamster Ubc9 homologue. The interactions between SC proteins and the Ubc9 protein may be significant for SC disassembly, which coincides with the repulsion of homologs by late prophase I, and also for the termination of sister centromere cohesiveness at anaphase II. Images PMID:9285814

  17. Small molecules that target phosphorylation dependent protein-protein interaction.

    PubMed

    Watanabe, Nobumoto; Osada, Hiroyuki

    2016-08-01

    Protein-protein interaction is one of the key events in the signal transduction pathway. The interaction changes the conformations, activities, localization and stabilities of the proteins, and transduces the signal to the next step. Frequently, this interaction occurs upon the protein phosphorylation. When upstream signals are stimulated, protein kinase(s) is/are activated and phosphorylate(s) their substrates, and induce the phosphorylation dependent protein-protein interaction. For this interaction, several domains in proteins are known to specifically recognize the phosphorylated residues of target proteins. These specific domains for interaction are important in the progression of the diseases caused by disordered signal transduction such as cancer. Thus small molecules that modulate this interaction are attractive lead compounds for the treatment of such diseases. In this review, we focused on three examples of phosphorylation dependent protein-protein interaction modules (14-3-3, polo box domain of Plk1 and F-box proteins in SCF ubiquitin ligases) and summarize small molecules that modulate their interaction. We also introduce our original screening system to identify such small molecules.

  18. Ontology integration to identify protein complex in protein interaction networks

    PubMed Central

    2011-01-01

    Background Protein complexes can be identified from the protein interaction networks derived from experimental data sets. However, these analyses are challenging because of the presence of unreliable interactions and the complex connectivity of the network. The integration of protein-protein interactions with the data from other sources can be leveraged for improving the effectiveness of protein complexes detection algorithms. Methods We have developed novel semantic similarity method, which use Gene Ontology (GO) annotations to measure the reliability of protein-protein interactions. The protein interaction networks can be converted into a weighted graph representation by assigning the reliability values to each interaction as a weight. Following the approach of that of the previously proposed clustering algorithm IPCA which expands clusters starting from seeded vertices, we present a clustering algorithm OIIP based on the new weighted Protein-Protein interaction networks for identifying protein complexes. Results The algorithm OIIP is applied to the protein interaction network of Sacchromyces cerevisiae and identifies many well known complexes. Experimental results show that the algorithm OIIP has higher F-measure and accuracy compared to other competing approaches. PMID:22165991

  19. Stabilized polyacrylic saccharide protein conjugates

    DOEpatents

    Callstrom, M.R.; Bednarski, M.D.; Gruber, P.R.

    1996-02-20

    This invention is directed to water soluble protein polymer conjugates which are stable in hostile environments. The conjugate comprises a protein which is linked to an acrylic polymer at multiple points through saccharide linker groups. 16 figs.

  20. Sorting sweet sorting. Protein secretion.

    PubMed

    Ponnambalam, S; Banting, G

    1996-09-01

    Membrane-spanning, lectin-like proteins in the eukaryotic secretory pathway seem to operate quality-control checkpoints by fine tuning protein exit or retention within each subcompartment. PMID:8805362

  1. Controlling allosteric networks in proteins

    NASA Astrophysics Data System (ADS)

    Dokholyan, Nikolay

    2013-03-01

    We present a novel methodology based on graph theory and discrete molecular dynamics simulations for delineating allosteric pathways in proteins. We use this methodology to uncover the structural mechanisms responsible for coupling of distal sites on proteins and utilize it for allosteric modulation of proteins. We will present examples where inference of allosteric networks and its rewiring allows us to ``rescue'' cystic fibrosis transmembrane conductance regulator (CFTR), a protein associated with fatal genetic disease cystic fibrosis. We also use our methodology to control protein function allosterically. We design a novel protein domain that can be inserted into identified allosteric site of target protein. Using a drug that binds to our domain, we alter the function of the target protein. We successfully tested this methodology in vitro, in living cells and in zebrafish. We further demonstrate transferability of our allosteric modulation methodology to other systems and extend it to become ligh-activatable.

  2. Leptospira Protein Expression During Infection

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We are characterizing protein expression in vivo during experimental leptospirosis using immunofluorescence microscopy. Coding regions for several proteins were identified through analysis of Leptospira interrogans serovar Copenhageni and L. borgpetersenii serovar Hardjo genomes. In addition, codi...

  3. Stabilized polyacrylic saccharide protein conjugates

    DOEpatents

    Callstrom, Matthew R.; Bednarski, Mark D.; Gruber, Patrick R.

    1996-01-01

    This invention is directed to water soluble protein polymer conjugates which are stabile in hostile environments. The conjugate comprises a protein which is linked to an acrylic polymer at multiple points through saccharide linker groups.

  4. Microtubules, Tubulins and Associated Proteins.

    ERIC Educational Resources Information Center

    Raxworthy, Michael J.

    1988-01-01

    Reviews much of what is known about microtubules, which are biopolymers consisting predominantly of subunits of the globular protein, tubulin. Describes the functions of microtubules, their structure and assembly, microtube associated proteins, and microtubule-disrupting agents. (TW)

  5. The cell-binding domain of intimin from enteropathogenic Escherichia coli binds to beta1 integrins.

    PubMed

    Frankel, G; Lider, O; Hershkoviz, R; Mould, A P; Kachalsky, S G; Candy, D C; Cahalon, L; Humphries, M J; Dougan, G

    1996-08-23

    Bacteria interact with mammalian cells surface molecules, such as integrins, to colonize tissues and evade immunological detection. Herein, the ability of intimin, an outer membrane protein from enteropathogenic Escherichia coli, to bind beta1 integrins was investigated. Solid-phase binding assays revealed binding of the carboxyl-terminal 280 amino acids of intimin (Int280) to alpha4beta1 and alpha5beta1 integrins. The binding required divalent ions (in particular, it was enhanced by Mn2+) and was inhibited by an RGD-containing peptide. Nonderivatized Int280, but not Int280CS (like Int280 but with Cys-937 replaced by Ser) blocked the binding of biotinylated Int280 to integrins. Int280 did not efficiently inhibit beta1 integrin binding of invasin from Yersinia pseudotuberculosis. Both intimin and invasin, immobilized on plastic surfaces, mediated adherence of resting or phorbol 12-myristate 13-acetate-activated human CD4(+) T cells, whereas fibronectin mediated the adherence of only activated T cells. T cell binding to intimin and invasin was integrin mediated because it was specifically blocked by an RGD-containing peptide and by antibodies directed against the integrin subunits beta1, alpha4, and alpha5. These results demonstrate a specific integrin binding activity for intimin that is related to, but distinct from, that of invasin. PMID:8702771

  6. Nanobiotechnology: protein-nanomaterial interactions.

    PubMed

    Kane, Ravi S; Stroock, Abraham D

    2007-01-01

    We review recent research that involves the interaction of nanomaterials such as nanoparticles, nanowires, and carbon nanotubes with proteins. We begin with a focus on the fundamentals of the structure and function of proteins on nanomaterials. We then review work in three areas that exploit these interactions: (1) sensing, (2) assembly of nanomaterials by proteins and proteins by nanomaterials, and (3) interactions with cells. We conclude with the identification of challenges and opportunities for the future. PMID:17335286

  7. The Papillomavirus E2 Proteins

    PubMed Central

    McBride, Alison A.

    2013-01-01

    The papillomavirus E2 proteins are pivotal to the viral life cycle and have well characterized functions in transcriptional regulation, initiation of DNA replication and partitioning the viral genome. The E2 proteins also function in vegetative DNA replication, post-transcriptional processes and possibly packaging. This review describes structural and functional aspects of the E2 proteins and their binding sites on the viral genome. It is intended to be a reference guide to this viral protein. PMID:23849793

  8. Tyrosine phosphorylation of WW proteins

    PubMed Central

    Reuven, Nina; Shanzer, Matan

    2015-01-01

    A number of key regulatory proteins contain one or two copies of the WW domain known to mediate protein–protein interaction via proline-rich motifs, such as PPxY. The Hippo pathway components take advantage of this module to transduce tumor suppressor signaling. It is becoming evident that tyrosine phosphorylation is a critical regulator of the WW proteins. Here, we review the current knowledge on the involved tyrosine kinases and their roles in regulating the WW proteins. PMID:25627656

  9. Endogenous protein phosphorylation and protein kinase activity in winged bean.

    PubMed

    Mukhopadhyay, K; Singh, M

    1997-10-01

    In winged bean (Psophocarpus tetragonolobus) protein kinases (E.C. 2.7.1.37) were found in all tissues studied. There was a significant increase in kinase activity during seed development, with a concomitant enhancement in the phosphorylation of a number of polypeptides; this was reversed in germinating seed cotyledons. Protein phosphorylation was apparently correlated with the increase in the protein content of the developing seed and the growing axis. At least three distinct autophosphorylating proteins could be distinguished in the developing seeds after SDS-PAGE, indicating the presence of different types of protein kinases in winged bean.

  10. Protein Adsorption in Three Dimensions

    PubMed Central

    Vogler, Erwin A.

    2011-01-01

    Recent experimental and theoretical work clarifying the physical chemistry of blood-protein adsorption from aqueous-buffer solution to various kinds of surfaces is reviewed and interpreted within the context of biomaterial applications, especially toward development of cardiovascular biomaterials. The importance of this subject in biomaterials surface science is emphasized by reducing the “protein-adsorption problem” to three core questions that require quantitative answer. An overview of the protein-adsorption literature identifies some of the sources of inconsistency among many investigators participating in more than five decades of focused research. A tutorial on the fundamental biophysical chemistry of protein adsorption sets the stage for a detailed discussion of the kinetics and thermodynamics of protein adsorption, including adsorption competition between two proteins for the same adsorbent immersed in a binary-protein mixture. Both kinetics and steady-state adsorption can be rationalized using a single interpretive paradigm asserting that protein molecules partition from solution into a three-dimensional (3D) interphase separating bulk solution from the physical-adsorbent surface. Adsorbed protein collects in one-or-more adsorbed layers, depending on protein size, solution concentration, and adsorbent surface energy (water wettability). The adsorption process begins with the hydration of an adsorbent surface brought into contact with an aqueous-protein solution. Surface hydration reactions instantaneously form a thin, pseudo-2D interface between the adsorbent and protein solution. Protein molecules rapidly diffuse into this newly-formed interface, creating a truly 3D interphase that inflates with arriving proteins and fills to capacity within milliseconds at mg/mL bulk-solution concentrations CB. This inflated interphase subsequently undergoes time-dependent (minutes-to-hours) decrease in volume VI by expulsion of either-or-both interphase water and

  11. Implication of Terminal Residues at Protein-Protein and Protein-DNA Interfaces.

    PubMed

    Martin, Olivier M F; Etheve, Loïc; Launay, Guillaume; Martin, Juliette

    2016-01-01

    Terminal residues of protein chains are charged and more flexible than other residues since they are constrained only on one side. Do they play a particular role in protein-protein and protein-DNA interfaces? To answer this question, we considered large sets of non-redundant protein-protein and protein-DNA complexes and analyzed the status of terminal residues and their involvement in interfaces. In protein-protein complexes, we found that more than half of terminal residues (62%) are either modified by attachment of a tag peptide (10%) or have missing coordinates in the analyzed structures (52%). Terminal residues are almost exclusively located at the surface of proteins (94%). Contrary to charged residues, they are not over or under-represented in protein-protein interfaces, but strongly prefer the peripheral region of interfaces when present at the interface (83% of terminal residues). The almost exclusive location of terminal residues at the surface of the proteins or in the rim regions of interfaces explains that experimental methods relying on tail hybridization can be successfully applied without disrupting the complexes under study. Concerning conformational rearrangement in protein-protein complexes, despite their expected flexibility, terminal residues adopt similar locations between the free and bound forms of the docking benchmark. In protein-DNA complexes, N-terminal residues are twice more frequent than C-terminal residues at interfaces. Both N-terminal and C-terminal residues are under-represented in interfaces, in contrast to positively charged residues, which are strongly favored. When located in protein-DNA interfaces, terminal residues prefer the periphery. N-terminal and C-terminal residues thus have particular properties with regard to interfaces, which cannot be reduced to their charged nature. PMID:27611671

  12. Implication of Terminal Residues at Protein-Protein and Protein-DNA Interfaces

    PubMed Central

    Martin, Olivier M. F.; Etheve, Loïc; Launay, Guillaume

    2016-01-01

    Terminal residues of protein chains are charged and more flexible than other residues since they are constrained only on one side. Do they play a particular role in protein-protein and protein-DNA interfaces? To answer this question, we considered large sets of non-redundant protein-protein and protein-DNA complexes and analyzed the status of terminal residues and their involvement in interfaces. In protein-protein complexes, we found that more than half of terminal residues (62%) are either modified by attachment of a tag peptide (10%) or have missing coordinates in the analyzed structures (52%). Terminal residues are almost exclusively located at the surface of proteins (94%). Contrary to charged residues, they are not over or under-represented in protein-protein interfaces, but strongly prefer the peripheral region of interfaces when present at the interface (83% of terminal residues). The almost exclusive location of terminal residues at the surface of the proteins or in the rim regions of interfaces explains that experimental methods relying on tail hybridization can be successfully applied without disrupting the complexes under study. Concerning conformational rearrangement in protein-protein complexes, despite their expected flexibility, terminal residues adopt similar locations between the free and bound forms of the docking benchmark. In protein-DNA complexes, N-terminal residues are twice more frequent than C-terminal residues at interfaces. Both N-terminal and C-terminal residues are under-represented in interfaces, in contrast to positively charged residues, which are strongly favored. When located in protein-DNA interfaces, terminal residues prefer the periphery. N-terminal and C-terminal residues thus have particular properties with regard to interfaces, which cannot be reduced to their charged nature. PMID:27611671

  13. Aeolotopic interactions of globular proteins

    PubMed Central

    Lomakin, Aleksey; Asherie, Neer; Benedek, George B.

    1999-01-01

    Protein crystallization, aggregation, liquid–liquid phase separation, and self-assembly are important in protein structure determination in the industrial processing of proteins and in the inhibition of protein condensation diseases. To fully describe such phase transformations in globular protein solutions, it is necessary to account for the strong spatial variation of the interactions on the protein surface. One difficulty is that each globular protein has its own unique surface, which is crucial for its biological function. However, the similarities amongst the macroscopic properties of different protein solutions suggest that there may exist a generic model that is capable of describing the nonuniform interactions between globular proteins. In this paper we present such a model, which includes the short-range interactions that vary from place to place on the surface of the protein. We show that this aeolotopic model [from the Greek aiolos (“variable”) and topos (“place”)] describes the phase diagram of globular proteins and provides insight into protein aggregation and crystallization. PMID:10449715

  14. Role of regulator of G protein signaling proteins in bone

    PubMed Central

    Keinan, David; Yang, Shuying; Cohen, Robert E.; Yuan, Xue; Liu, Tongjun; Li, Yi-Ping

    2014-01-01

    Regulators of G protein signaling (RGS) proteins are a family with more than 30 proteins that all contain an RGS domain. In the past decade, increasing evidence has indicated that RGS proteins play crucial roles in the regulation of G protein coupling receptors (GPCR), G proteins, and calcium signaling during cell proliferation, migration, and differentiation in a variety of tissues. In bone, those proteins modulate bone development and remodeling by influencing various signaling pathways such as GPCR-G protein signaling, Wnt, calcium oscillations and PTH. This review summarizes the recent advances in the understanding of the regulation of RGS genes expression, as well as the functions and mechanisms of RGS proteins, especially in regulating GPCR-G protein signaling, Wnt signaling, calcium oscillations signaling and PTH signaling during bone development and remodeling. This review also highlights the regulation of different RGS proteins in osteoblasts, chondrocytes and osteoclasts. The knowledge from the recent advances of RGS study summarized in the review would provide the insights into new therapies for bone diseases. PMID:24389209

  15. Highly specific protein-protein interactions, evolution and negative design.

    PubMed

    Sear, Richard P

    2004-12-01

    We consider highly specific protein-protein interactions in proteomes of simple model proteins. We are inspired by the work of Zarrinpar et al (2003 Nature 426 676). They took a binding domain in a signalling pathway in yeast and replaced it with domains of the same class but from different organisms. They found that the probability of a protein binding to a protein from the proteome of a different organism is rather high, around one half. We calculate the probability of a model protein from one proteome binding to the protein of a different proteome. These proteomes are obtained by sampling the space of functional proteomes uniformly. In agreement with Zarrinpar et al we find that the probability of a protein binding a protein from another proteome is rather high, of order one tenth. Our results, together with those of Zarrinpar et al, suggest that designing, say, a peptide to block or reconstitute a single signalling pathway, without affecting any other pathways, requires knowledge of all the partners of the class of binding domains the peptide is designed to mimic. This knowledge is required to use negative design to explicitly design out interactions of the peptide with proteins other than its target. We also found that patches that are required to bind with high specificity evolve more slowly than those that are required only to not bind to any other patch. This is consistent with some analysis of sequence data for proteins engaged in highly specific interactions.

  16. Biophysics of protein evolution and evolutionary protein biophysics

    PubMed Central

    Sikosek, Tobias; Chan, Hue Sun

    2014-01-01

    The study of molecular evolution at the level of protein-coding genes often entails comparing large datasets of sequences to infer their evolutionary relationships. Despite the importance of a protein's structure and conformational dynamics to its function and thus its fitness, common phylogenetic methods embody minimal biophysical knowledge of proteins. To underscore the biophysical constraints on natural selection, we survey effects of protein mutations, highlighting the physical basis for marginal stability of natural globular proteins and how requirement for kinetic stability and avoidance of misfolding and misinteractions might have affected protein evolution. The biophysical underpinnings of these effects have been addressed by models with an explicit coarse-grained spatial representation of the polypeptide chain. Sequence–structure mappings based on such models are powerful conceptual tools that rationalize mutational robustness, evolvability, epistasis, promiscuous function performed by ‘hidden’ conformational states, resolution of adaptive conflicts and conformational switches in the evolution from one protein fold to another. Recently, protein biophysics has been applied to derive more accurate evolutionary accounts of sequence data. Methods have also been developed to exploit sequence-based evolutionary information to predict biophysical behaviours of proteins. The success of these approaches demonstrates a deep synergy between the fields of protein biophysics and protein evolution. PMID:25165599

  17. Protein adsorption to hydrocephalus shunt catheters: CSF protein adsorption

    PubMed Central

    Brydon, H.; Keir, G.; Thompson, E.; Bayston, R.; Hayward, R.; Harkness, W.

    1998-01-01

    OBJECTIVE—To assess the quantity and nature of the proteins that adsorb to hydrocephalus shunt catheters after implantation, and to determine whether sufficient could accumulate to obstruct the catheter.
DESIGN—Elution of proteins from 102 explanted shunt catheters, with protein assay and electrophoresis of the eluate, and scanning electron microscopy (SEM) of the catheters.
RESULTS—The amount of protein elutable was extremely low, and significant protein, apart from a thin film, was not found on SEM. Qualitative analysis disclosed that most of the adsorbed protein was albumin.
CONCLUSIONS—Protein deposition on hydrocephalus catheters does not occur in sufficient quantities to cause catheter obstruction.

 PMID:9598681

  18. Flowering Buds of Globular Proteins: Transpiring Simplicity of Protein Organization

    PubMed Central

    Berezovsky, Igor N.

    2002-01-01

    Structural and functional complexity of proteins is dramatically reduced to a simple linear picture when the laws of polymer physics are considered. A basic unit of the protein structure is a nearly standard closed loop of 25–35 amino acid residues, and every globular protein is built of consecutively connected closed loops. The physical necessity of the closed loops had been apparently imposed on the early stages of protein evolution. Indeed, the most frequent prototype sequence motifs in prokaryotic proteins have the same sequence size, and their high match representatives are found as closed loops in crystallized proteins. Thus, the linear organization of the closed loop elements is a quintessence of protein evolution, structure and folding. PMID:18629251

  19. Commercial Protein Crystal Growth: Protein Crystallization Facility (CPCG-H)

    NASA Astrophysics Data System (ADS)

    DeLucas, Lawrence J.

    2002-12-01

    Within the human body, there are thousands of different proteins that serve a variety of different functions, such as making it possible for red blood cells to carry oxygen in our bodies. Yet proteins can also be involved in diseases. Each protein has a particular chemical structure, which means it has a unique shape. It is this three-dimensional shape that allows each protein to do its job by interacting with chemicals or binding with other proteins. If researchers can determine the shape, or shapes, of a protein, they can learn how it works. This information can then be used by the pharmaceutical industry to develop new drugs or improve the way medications work. The NASA Commercial Space Center sponsoring this experiment - the Center for Biophysical Sciences and Engineering at the University of Alabama at Birmingham - has more than 60 industry and academic partners who grow protein crystals and use the information in drug design projects.

  20. Protein subcellular localization assays using split fluorescent proteins

    DOEpatents

    Waldo, Geoffrey S.; Cabantous, Stephanie

    2009-09-08

    The invention provides protein subcellular localization assays using split fluorescent protein systems. The assays are conducted in living cells, do not require fixation and washing steps inherent in existing immunostaining and related techniques, and permit rapid, non-invasive, direct visualization of protein localization in living cells. The split fluorescent protein systems used in the practice of the invention generally comprise two or more self-complementing fragments of a fluorescent protein, such as GFP, wherein one or more of the fragments correspond to one or more beta-strand microdomains and are used to "tag" proteins of interest, and a complementary "assay" fragment of the fluorescent protein. Either or both of the fragments may be functionalized with a subcellular targeting sequence enabling it to be expressed in or directed to a particular subcellular compartment (i.e., the nucleus).

  1. Yeast protein-protein interaction assays and screens.

    PubMed

    de Folter, Stefan; Immink, Richard G H

    2011-01-01

    Most transcription factors fulfill their role in protein complexes. As a consequence, information about their interaction capacity sheds light on a protein's function and the molecular mechanism underlying this activity. The yeast two-hybrid GAL4 (Y2H) assay is a powerful method to unravel and identify the composition of protein complexes. This in vivo based system makes use of two functional protein domains of the GAL4 transcription factor, each fused to a protein of interest. Upon interaction between the two proteins under study, a transcriptional activator gets reconstituted and reporter genes get activated, allowing the yeast to grow on selective medium. In this chapter protocols are given for Y2H library screening, directed Y2H screening, Y2H matrix screening, and YnH screening involving more than two proteins. PMID:21720951

  2. Protein enriched pasta: structure and digestibility of its protein network.

    PubMed

    Laleg, Karima; Barron, Cécile; Santé-Lhoutellier, Véronique; Walrand, Stéphane; Micard, Valérie

    2016-02-01

    Wheat (W) pasta was enriched in 6% gluten (G), 35% faba (F) or 5% egg (E) to increase its protein content (13% to 17%). The impact of the enrichment on the multiscale structure of the pasta and on in vitro protein digestibility was studied. Increasing the protein content (W- vs. G-pasta) strengthened pasta structure at molecular and macroscopic scales but reduced its protein digestibility by 3% by forming a higher covalently linked protein network. Greater changes in the macroscopic and molecular structure of the pasta were obtained by varying the nature of protein used for enrichment. Proteins in G- and E-pasta were highly covalently linked (28-32%) resulting in a strong pasta structure. Conversely, F-protein (98% SDS-soluble) altered the pasta structure by diluting gluten and formed a weak protein network (18% covalent link). As a result, protein digestibility in F-pasta was significantly higher (46%) than in E- (44%) and G-pasta (39%). The effect of low (55 °C, LT) vs. very high temperature (90 °C, VHT) drying on the protein network structure and digestibility was shown to cause greater molecular changes than pasta formulation. Whatever the pasta, a general strengthening of its structure, a 33% to 47% increase in covalently linked proteins and a higher β-sheet structure were observed. However, these structural differences were evened out after the pasta was cooked, resulting in identical protein digestibility in LT and VHT pasta. Even after VHT drying, F-pasta had the best amino acid profile with the highest protein digestibility, proof of its nutritional interest.

  3. Protein enriched pasta: structure and digestibility of its protein network.

    PubMed

    Laleg, Karima; Barron, Cécile; Santé-Lhoutellier, Véronique; Walrand, Stéphane; Micard, Valérie

    2016-02-01

    Wheat (W) pasta was enriched in 6% gluten (G), 35% faba (F) or 5% egg (E) to increase its protein content (13% to 17%). The impact of the enrichment on the multiscale structure of the pasta and on in vitro protein digestibility was studied. Increasing the protein content (W- vs. G-pasta) strengthened pasta structure at molecular and macroscopic scales but reduced its protein digestibility by 3% by forming a higher covalently linked protein network. Greater changes in the macroscopic and molecular structure of the pasta were obtained by varying the nature of protein used for enrichment. Proteins in G- and E-pasta were highly covalently linked (28-32%) resulting in a strong pasta structure. Conversely, F-protein (98% SDS-soluble) altered the pasta structure by diluting gluten and formed a weak protein network (18% covalent link). As a result, protein digestibility in F-pasta was significantly higher (46%) than in E- (44%) and G-pasta (39%). The effect of low (55 °C, LT) vs. very high temperature (90 °C, VHT) drying on the protein network structure and digestibility was shown to cause greater molecular changes than pasta formulation. Whatever the pasta, a general strengthening of its structure, a 33% to 47% increase in covalently linked proteins and a higher β-sheet structure were observed. However, these structural differences were evened out after the pasta was cooked, resulting in identical protein digestibility in LT and VHT pasta. Even after VHT drying, F-pasta had the best amino acid profile with the highest protein digestibility, proof of its nutritional interest. PMID:26829164

  4. Tetramer formation in Arabidopsis MADS domain proteins: analysis of a protein-protein interaction network

    PubMed Central

    2014-01-01

    Background MADS domain proteins are transcription factors that coordinate several important developmental processes in plants. These proteins interact with other MADS domain proteins to form dimers, and it has been proposed that they are able to associate as tetrameric complexes that regulate transcription of target genes. Whether the formation of functional tetramers is a widespread property of plant MADS domain proteins, or it is specific to few of these transcriptional regulators remains unclear. Results We analyzed the structure of the network of physical interactions among MADS domain proteins in Arabidopsis thaliana. We determined the abundance of subgraphs that represent the connection pattern expected for a MADS domain protein heterotetramer. These subgraphs were significantly more abundant in the MADS domain protein interaction network than in randomized analogous networks. Importantly, these subgraphs are not significantly frequent in a protein interaction network of TCP plant transcription factors, when compared to expectation by chance. In addition, we found that MADS domain proteins in tetramer-like subgraphs are more likely to be expressed jointly than proteins in other subgraphs. This effect is mainly due to proteins in the monophyletic MIKC clade, as there is no association between tetramer-like subgraphs and co-expression for proteins outside this clade. Conclusions Our results support that the tendency to form functional tetramers is widespread in the MADS domain protein-protein interaction network. Our observations also suggest that this trend is prevalent, or perhaps exclusive, for proteins in the MIKC clade. Because it is possible to retrodict several experimental results from our analyses, our work can be an important aid to make new predictions and facilitates experimental research on plant MADS domain proteins. PMID:24468197

  5. Viruses and viral proteins

    PubMed Central

    Verdaguer, Nuria; Ferrero, Diego; Murthy, Mathur R. N.

    2014-01-01

    For more than 30 years X-ray crystallography has been by far the most powerful approach for determining the structures of viruses and viral proteins at atomic resolution. The information provided by these structures, which covers many important aspects of the viral life cycle such as cell-receptor recognition, viral entry, nucleic acid transfer and genome replication, has extensively enriched our vision of the virus world. Many of the structures available correspond to potential targets for antiviral drugs against important human pathogens. This article provides an overview of the current knowledge of different structural aspects of the above-mentioned processes. PMID:25485129

  6. SERUM PROTEIN PROFILES IN COCCIDIOIDOMYCOSIS

    PubMed Central

    Reed, William B.; Heiskell, Charles L.; Holeman, Charles W.; Carpenter, Charles

    1962-01-01

    Serum protein analysis is a valuable addition to the present methods for evaluating the status of the individual patient with coccidioidomycosis. The albumin protein and albumin glycoprotein decrease and gamma protein increases in relation to severity of infection. In 40 patients with coccidioidomycosis, changes in individual protein fractions could be significantly correlated with conventional laboratory tests, such as the complement fixation test, erythrocyte sedimentation rate and hematocrit. Changes in the alpha, glycoprotein concentration, the erythrocyte sedimentation rate and the hematocrit value appear to be related to the degree of inflammation, while the changes in the gamma protein and the beta, glycoprotein appear to be related to the specific antibody response. PMID:13973566

  7. Serum protein profiles in coccidioidomycosis.

    PubMed

    REED, W B; HEISKELL, C L; HOLEMAN, C W; CARPENTER, C

    1962-12-01

    Serum protein analysis is a valuable addition to the present methods for evaluating the status of the individual patient with coccidioidomycosis. The albumin protein and albumin glycoprotein decrease and gamma protein increases in relation to severity of infection. In 40 patients with coccidioidomycosis, changes in individual protein fractions could be significantly correlated with conventional laboratory tests, such as the complement fixation test, erythrocyte sedimentation rate and hematocrit. Changes in the alpha, glycoprotein concentration, the erythrocyte sedimentation rate and the hematocrit value appear to be related to the degree of inflammation, while the changes in the gamma protein and the beta, glycoprotein appear to be related to the specific antibody response.

  8. Probing High-density Functional Protein Microarrays to Detect Protein-protein Interactions.

    PubMed

    Fasolo, Joseph; Im, Hogune; Snyder, Michael P

    2015-01-01

    High-density functional protein microarrays containing ~4,200 recombinant yeast proteins are examined for kinase protein-protein interactions using an affinity purified yeast kinase fusion protein containing a V5-epitope tag for read-out. Purified kinase is obtained through culture of a yeast strain optimized for high copy protein production harboring a plasmid containing a Kinase-V5 fusion construct under a GAL inducible promoter. The yeast is grown in restrictive media with a neutral carbon source for 6 hr followed by induction with 2% galactose. Next, the culture is harvested and kinase is purified using standard affinity chromatographic techniques to obtain a highly purified protein kinase for use in the assay. The purified kinase is diluted with kinase buffer to an appropriate range for the assay and the protein microarrays are blocked prior to hybridization with the protein microarray. After the hybridization, the arrays are probed with monoclonal V5 antibody to identify proteins bound by the kinase-V5 protein. Finally, the arrays are scanned using a standard microarray scanner, and data is extracted for downstream informatics analysis to determine a high confidence set of protein interactions for downstream validation in vivo. PMID:26274875

  9. Protein-protein interaction network-based detection of functionally similar proteins within species.

    PubMed

    Song, Baoxing; Wang, Fen; Guo, Yang; Sang, Qing; Liu, Min; Li, Dengyun; Fang, Wei; Zhang, Deli

    2012-07-01

    Although functionally similar proteins across species have been widely studied, functionally similar proteins within species showing low sequence similarity have not been examined in detail. Identification of these proteins is of significant importance for understanding biological functions, evolution of protein families, progression of co-evolution, and convergent evolution and others which cannot be obtained by detection of functionally similar proteins across species. Here, we explored a method of detecting functionally similar proteins within species based on graph theory. After denoting protein-protein interaction networks using graphs, we split the graphs into subgraphs using the 1-hop method. Proteins with functional similarities in a species were detected using a method of modified shortest path to compare these subgraphs and to find the eligible optimal results. Using seven protein-protein interaction networks and this method, some functionally similar proteins with low sequence similarity that cannot detected by sequence alignment were identified. By analyzing the results, we found that, sometimes, it is difficult to separate homologous from convergent evolution. Evaluation of the performance of our method by gene ontology term overlap showed that the precision of our method was excellent.

  10. Intrinsic Localized Modes in Proteins

    PubMed Central

    Nicolaï, Adrien; Delarue, Patrice; Senet, Patrick

    2015-01-01

    Protein dynamics is essential for proteins to function. Here we predicted the existence of rare, large nonlinear excitations, termed intrinsic localized modes (ILMs), of the main chain of proteins based on all-atom molecular dynamics simulations of two fast-folder proteins and of a rigid α/β protein at 300 K and at 380 K in solution. These nonlinear excitations arise from the anharmonicity of the protein dynamics. The ILMs were detected by computing the Shannon entropy of the protein main-chain fluctuations. In the non-native state (significantly explored at 380 K), the probability of their excitation was increased by a factor between 9 and 28 for the fast-folder proteins and by a factor 2 for the rigid protein. This enhancement in the non-native state was due to glycine, as demonstrated by simulations in which glycine was mutated to alanine. These ILMs might play a functional role in the flexible regions of proteins and in proteins in a non-native state (i.e. misfolded or unfolded states). PMID:26658321

  11. Protein crystal growth in microgravity

    NASA Technical Reports Server (NTRS)

    Rosenblum, William M.; Delucas, Lawrence J.; Wilson, William W.

    1989-01-01

    Major advances have been made in several of the experimental aspects of protein crystallography, leaving protein crystallization as one of the few remaining bottlenecks. As a result, it has become important that the science of protein crystal growth is better understood and that improved methods for protein crystallization are developed. Preliminary experiments with both small molecules and proteins indicate that microgravity may beneficially affect crystal growth. For this reason, a series of protein crystal growth experiments using the Space Shuttle was initiated. The preliminary space experiments were used to evolve prototype hardware that will form the basis for a more advanced system that can be used to evaluate effects of gravity on protein crystal growth. Various optical techniques are being utilized to monitor the crystal growth process from the incipient or nucleation stage and throughout the growth phase. The eventual goal of these studies is to develop a system which utilizes optical monitoring for dynamic control of the crystallization process.

  12. Protein Adsorption in Three Dimensions

    PubMed Central

    Vogler, Erwin A.

    2011-01-01

    Recent experimental and theoretical work clarifying the physical chemistry of blood-protein adsorption from aqueous-buffer solution to various kinds of surfaces is reviewed and interpreted within the context of biomaterial applications, especially toward development of cardiovascular biomaterials. The importance of this subject in biomaterials surface science is emphasized by reducing the “protein-adsorption problem” to three core questions that require quantitative answer. An overview of the protein-adsorption literature identifies some of the sources of inconsistency among many investigators participating in more than five decades of focused research. A tutorial on the fundamental biophysical chemistry of protein adsorption sets the stage for a detailed discussion of the kinetics and thermodynamics of protein adsorption, including adsorption competition between two proteins for the same adsorbent immersed in a binary-protein mixture. Both kinetics and steady-state adsorption can be rationalized using a single interpretive paradigm asserting that protein molecules partition from solution into a three-dimensional (3D) interphase separating bulk solution from the physical-adsorbent surface. Adsorbed protein collects in one-or-more adsorbed layers, depending on protein size, solution concentration, and adsorbent surface energy (water wettability). The adsorption process begins with the hydration of an adsorbent surface brought into contact with an aqueous-protein solution. Surface hydration reactions instantaneously form a thin, pseudo-2D interface between the adsorbent and protein solution. Protein molecules rapidly diffuse into this newly-formed interface, creating a truly 3D interphase that inflates with arriving proteins and fills to capacity within milliseconds at mg/mL bulk-solution concentrations CB. This inflated interphase subsequently undergoes time-dependent (minutes-to-hours) decrease in volume VI by expulsion of either-or-both interphase water and

  13. Protein Repeats from First Principles.

    PubMed

    Turjanski, Pablo; Parra, R Gonzalo; Espada, Rocío; Becher, Verónica; Ferreiro, Diego U

    2016-01-01

    Some natural proteins display recurrent structural patterns. Despite being highly similar at the tertiary structure level, repeating patterns within a single repeat protein can be extremely variable at the sequence level. We use a mathematical definition of a repetition and investigate the occurrences of these in sequences of different protein families. We found that long stretches of perfect repetitions are infrequent in individual natural proteins, even for those which are known to fold into structures of recurrent structural motifs. We found that natural repeat proteins are indeed repetitive in their families, exhibiting abundant stretches of 6 amino acids or longer that are perfect repetitions in the reference family. We provide a systematic quantification for this repetitiveness. We show that this form of repetitiveness is not exclusive of repeat proteins, but also occurs in globular domains. A by-product of this work is a fast quantification of the likelihood of a protein to belong to a family. PMID:27044676

  14. Mathematical methods for protein science

    SciTech Connect

    Hart, W.; Istrail, S.; Atkins, J.

    1997-12-31

    Understanding the structure and function of proteins is a fundamental endeavor in molecular biology. Currently, over 100,000 protein sequences have been determined by experimental methods. The three dimensional structure of the protein determines its function, but there are currently less than 4,000 structures known to atomic resolution. Accordingly, techniques to predict protein structure from sequence have an important role in aiding the understanding of the Genome and the effects of mutations in genetic disease. The authors describe current efforts at Sandia to better understand the structure of proteins through rigorous mathematical analyses of simple lattice models. The efforts have focused on two aspects of protein science: mathematical structure prediction, and inverse protein folding.

  15. The Papillomavirus E2 proteins

    SciTech Connect

    McBride, Alison A.

    2013-10-15

    The papillomavirus E2 proteins are pivotal to the viral life cycle and have well characterized functions in transcriptional regulation, initiation of DNA replication and partitioning the viral genome. The E2 proteins also function in vegetative DNA replication, post-transcriptional processes and possibly packaging. This review describes structural and functional aspects of the E2 proteins and their binding sites on the viral genome. It is intended to be a reference guide to this viral protein. - Highlights: • Overview of E2 protein functions. • Structural domains of the papillomavirus E2 proteins. • Analysis of E2 binding sites in different genera of papillomaviruses. • Compilation of E2 associated proteins. • Comparison of key mutations in distinct E2 functions.

  16. Dietary protein and blood pressure.

    PubMed

    Bursztyn, P G; Vas Dias, F W

    1985-01-01

    Vegetarians have lower blood pressures than omnivores. Dietary protein may be partly responsible. Human volunteers, whose normal diet contained little soya protein, were given soya based foods to replace some of the meat in their diet. During this period over 20% of the total protein intake was derived from soya, however blood pressures remained unchanged. Rabbits were given diets based on either soya, casein, or fish protein. The animals' diets were then changed to one of the other protein sources. During the subsequent 3 weeks, small increases in blood pressure were seen in the casein and soya groups. When rabbits were given fat enriched diets, blood pressures rose but the increase was independent of the type of protein in the diet. It is concluded that the type of protein consumed is unlikely to account for the blood pressure differences between vegetarians and omnivores. Arguments are presented suggesting that other dietary components, such as fat or fibre may be responsible.

  17. Protein Repeats from First Principles.

    PubMed

    Turjanski, Pablo; Parra, R Gonzalo; Espada, Rocío; Becher, Verónica; Ferreiro, Diego U

    2016-04-05

    Some natural proteins display recurrent structural patterns. Despite being highly similar at the tertiary structure level, repeating patterns within a single repeat protein can be extremely variable at the sequence level. We use a mathematical definition of a repetition and investigate the occurrences of these in sequences of different protein families. We found that long stretches of perfect repetitions are infrequent in individual natural proteins, even for those which are known to fold into structures of recurrent structural motifs. We found that natural repeat proteins are indeed repetitive in their families, exhibiting abundant stretches of 6 amino acids or longer that are perfect repetitions in the reference family. We provide a systematic quantification for this repetitiveness. We show that this form of repetitiveness is not exclusive of repeat proteins, but also occurs in globular domains. A by-product of this work is a fast quantification of the likelihood of a protein to belong to a family.

  18. Protein Repeats from First Principles

    PubMed Central

    Turjanski, Pablo; Parra, R. Gonzalo; Espada, Rocío; Becher, Verónica; Ferreiro, Diego U.

    2016-01-01

    Some natural proteins display recurrent structural patterns. Despite being highly similar at the tertiary structure level, repeating patterns within a single repeat protein can be extremely variable at the sequence level. We use a mathematical definition of a repetition and investigate the occurrences of these in sequences of different protein families. We found that long stretches of perfect repetitions are infrequent in individual natural proteins, even for those which are known to fold into structures of recurrent structural motifs. We found that natural repeat proteins are indeed repetitive in their families, exhibiting abundant stretches of 6 amino acids or longer that are perfect repetitions in the reference family. We provide a systematic quantification for this repetitiveness. We show that this form of repetitiveness is not exclusive of repeat proteins, but also occurs in globular domains. A by-product of this work is a fast quantification of the likelihood of a protein to belong to a family. PMID:27044676

  19. Protein-protein interaction network analysis of cirrhosis liver disease

    PubMed Central

    Safaei, Akram; Rezaei Tavirani, Mostafa; Arefi Oskouei, Afsaneh; Zamanian Azodi, Mona; Mohebbi, Seyed Reza; Nikzamir, Abdol Rahim

    2016-01-01

    Aim: Evaluation of biological characteristics of 13 identified proteins of patients with cirrhotic liver disease is the main aim of this research. Background: In clinical usage, liver biopsy remains the gold standard for diagnosis of hepatic fibrosis. Evaluation and confirmation of liver fibrosis stages and severity of chronic diseases require a precise and noninvasive biomarkers. Since the early detection of cirrhosis is a clinical problem, achieving a sensitive, specific and predictive novel method based on biomarkers is an important task. Methods: Essential analysis, such as gene ontology (GO) enrichment and protein-protein interactions (PPI) was undergone EXPASy, STRING Database and DAVID Bioinformatics Resources query. Results: Based on GO analysis, most of proteins are located in the endoplasmic reticulum lumen, intracellular organelle lumen, membrane-enclosed lumen, and extracellular region. The relevant molecular functions are actin binding, metal ion binding, cation binding and ion binding. Cell adhesion, biological adhesion, cellular amino acid derivative, metabolic process and homeostatic process are the related processes. Protein-protein interaction network analysis introduced five proteins (fibroblast growth factor receptor 4, tropomyosin 4, tropomyosin 2 (beta), lectin, Lectin galactoside-binding soluble 3 binding protein and apolipoprotein A-I) as hub and bottleneck proteins. Conclusion: Our result indicates that regulation of lipid metabolism and cell survival are important biological processes involved in cirrhosis disease. More investigation of above mentioned proteins will provide a better understanding of cirrhosis disease. PMID:27099671

  20. A new protein structure representation for efficient protein function prediction.

    PubMed

    Maghawry, Huda A; Mostafa, Mostafa G M; Gharib, Tarek F

    2014-12-01

    One of the challenging problems in bioinformatics is the prediction of protein function. Protein function is the main key that can be used to classify different proteins. Protein function can be inferred experimentally with very small throughput or computationally with very high throughput. Computational methods are sequence based or structure based. Structure-based methods produce more accurate protein function prediction. In this article, we propose a new protein structure representation for efficient protein function prediction. The representation is based on three-dimensional patterns of protein residues. In the analysis, we used protein function based on enzyme activity through six mechanistically diverse enzyme superfamilies: amidohydrolase, crotonase, haloacid dehalogenase, isoprenoid synthase type I, and vicinal oxygen chelate. We applied three different classification methods, naïve Bayes, k-nearest neighbors, and random forest, to predict the enzyme superfamily of a given protein. The prediction accuracy using the proposed representation outperforms a recently introduced representation method that is based only on the distance patterns. The results show that the proposed representation achieved prediction accuracy up to 98%, with improvement of about 10% on average.

  1. Converting a marginally hydrophobic soluble protein into a membrane protein.

    PubMed

    Nørholm, Morten H H; Cunningham, Fiona; Deber, Charles M; von Heijne, Gunnar

    2011-03-18

    δ-Helices are marginally hydrophobic α-helical segments in soluble proteins that exhibit certain sequence characteristics of transmembrane (TM) helices [Cunningham, F., Rath, A., Johnson, R. M. & Deber, C. M. (2009). Distinctions between hydrophobic helices in globular proteins and TM segments as factors in protein sorting. J. Biol. Chem., 284, 5395-402]. In order to better understand the difference between δ-helices and TM helices, we have studied the insertion of five TM-like δ-helices into dog pancreas microsomal membranes. Using model constructs in which an isolated δ-helix is engineered into a bona fide membrane protein, we find that, for two δ-helices originating from secreted proteins, at least three single-nucleotide mutations are necessary to obtain efficient membrane insertion, whereas one mutation is sufficient in a δ-helix from the cytosolic protein P450BM-3. We further find that only when the entire upstream region of the mutated δ-helix in the intact cytochrome P450BM-3 is deleted does a small fraction of the truncated protein insert into microsomes. Our results suggest that upstream portions of the polypeptide, as well as embedded charged residues, protect δ-helices in globular proteins from being recognized by the signal recognition particle-Sec61 endoplasmic-reticulum-targeting machinery and that δ-helices in secreted proteins are mutationally more distant from TM helices than δ-helices in cytosolic proteins.

  2. Optimization of the electrostatic interactions in protein-protein complexes

    NASA Astrophysics Data System (ADS)

    Alexov, Emil; Brock, Kelly; Kundrotas, Petras

    2007-03-01

    Electrostatic energy is one of the driving forces of protein-protein association. Understanding the role of the energy components on the energetics of protein-protein association will help us in engineering protein-protein interactions and could lead to development of scoring functions that can rank alternative models and decoys. Here we investigate whether the components of the electrostatic energy of protein-protein complexes is optimized in respect to random distribution of the charged residues. We report a clear tendency that coulombic electrostatic interactions are optimized, while the reaction field energy is inversely optimized. It was found that the maximum of the coulombic energy Z-score is shifted 3 units away from the origin and the maximum of the reaction field energy by 2 units. Such a large shift of the Z-score of both coulombic and reaction field energies indicates that wild-type protein-protein interactions are in most cases optimized in terms of coulombic interactions while compromising reaction field energy. Based on these finding a scoring function was developed as a linear combination of the Z-score of the coulombic interactions minus Z-score of the reaction field energy. The scoring function was tested against the decoy sets and it was shown that in majority of the cases we can identify the wild-type complex among hundreds of decoys.

  3. Protein farnesyltransferase and protein prenylation in Plasmodium falciparum.

    PubMed

    Chakrabarti, Debopam; Da Silva, Thiago; Barger, Jennifer; Paquette, Steve; Patel, Hetal; Patterson, Shelley; Allen, Charles M

    2002-11-01

    Comparison of the malaria parasite and mammalian protein prenyltransferases and their cellular substrates is important for establishing this enzyme as a target for developing antimalarial agents. Nineteen heptapeptides differing only in their carboxyl-terminal amino acid were tested as alternative substrates of partially purified Plasmodium falciparum protein farnesyltransferase. Only NRSCAIM and NRSCAIQ serve as substrates, with NRSCAIM being the best. Peptidomimetics, FTI-276 and GGTI-287, inhibit the transferase with IC(50) values of 1 and 32 nm, respectively. Incubation of P. falciparum-infected erythrocytes with [(3)H]farnesol labels 50- and 22-28-kDa proteins, whereas [(3)H]geranylgeraniol labels only 22-28-kDa proteins. The 50-kDa protein is shown to be farnesylated, whereas the 22-28-kDa proteins are geranylgeranylated, irrespective of the labeling prenol. Protein labeling is inhibited more than 50% by either 5 microm FTI-277 or GGTI-298. The same concentration of inhibitors also inhibits parasite growth from the ring stage by 50%, decreases expression of prenylated proteins as measured with prenyl-specific antibody, and inhibits parasite differentiation beyond the trophozoite stage. Furthermore, differentiation specific prenylation of P. falciparum proteins is demonstrated. Protein labeling is detected predominantly during the trophozoite to schizont and schizont to ring transitions. These results demonstrate unique properties of protein prenylation in P. falciparum: a limited specificity of the farnesyltransferase for peptide substrates compared with mammalian enzymes, the ability to use farnesol to label both farnesyl and geranylgeranyl moieties on proteins, differentiation specific protein prenylation, and the ability of peptidomimetic prenyltransferase inhibitors to block parasite differentiation.

  4. Introduction to protein crystallization.

    PubMed

    McPherson, Alexander; Gavira, Jose A

    2014-01-01

    Protein crystallization was discovered by chance about 150 years ago and was developed in the late 19th century as a powerful purification tool and as a demonstration of chemical purity. The crystallization of proteins, nucleic acids and large biological complexes, such as viruses, depends on the creation of a solution that is supersaturated in the macromolecule but exhibits conditions that do not significantly perturb its natural state. Supersaturation is produced through the addition of mild precipitating agents such as neutral salts or polymers, and by the manipulation of various parameters that include temperature, ionic strength and pH. Also important in the crystallization process are factors that can affect the structural state of the macromolecule, such as metal ions, inhibitors, cofactors or other conventional small molecules. A variety of approaches have been developed that combine the spectrum of factors that effect and promote crystallization, and among the most widely used are vapor diffusion, dialysis, batch and liquid-liquid diffusion. Successes in macromolecular crystallization have multiplied rapidly in recent years owing to the advent of practical, easy-to-use screening kits and the application of laboratory robotics. A brief review will be given here of the most popular methods, some guiding principles and an overview of current technologies.

  5. Cow's Milk Protein Allergy.

    PubMed

    Mousan, Grace; Kamat, Deepak

    2016-10-01

    Cow's milk protein allergy (CMPA) is a common condition encountered in children with incidence estimated as 2% to 7.5% in the first year of life. Formula and breast-fed babies can present with symptoms of CMPA. It is important to accurately diagnose CMPA to avoid the consequences of either under- or overdiagnosis. CMPA is classically categorized into immunoglobulin E (IgE)- or non-IgE-mediated reaction that vary in clinical manifestations, diagnostic evaluation, and prognosis. The most commonly involved systems in patients with CMPA are gastrointestinal, skin, and respiratory. Evaluation of CMPA starts with good data gathering followed by testing if indicated. Treatment is simply by avoidance of cow's milk protein (CMP) in the child's or mother's diet, if exclusively breast-feeding. This article reviews the definition, epidemiology, risk factors, pathogenesis, clinical presentation, evaluation, management, and prognosis of CMPA and provides an overview of different options for formulas and their indication in the treatment of CMPA. PMID:27582492

  6. Rat myocardial protein degradation.

    PubMed

    Steer, J H; Hopkins, B E

    1981-07-01

    1. Myocardial protein degradation rates were determined by following tyrosine release from rat isolated left hemi-atria in vitro. 2. After two 20 min preincubations the rate of tyrosine release from hemi-atria was constant for 4 h. 3. Skeletal muscle protein degradation was determined by following tyrosine release from rat isolated hemi-diaphragm (Fulks, Li & Goldberg, 1975). 4. Insulin (10(-7) M) inhibited tyrosine release from hemi-atria and hemi-diaphragm to a similar extent. A 48 h fast increased tyrosine release rate from hemi-diaphragm and decreased tyrosine release rate from hemi-atria. Hemi-diaphragm tyrosine release was inhibited by 15 mmol/l D-glucose but a variety of concentrations of D-glucose (0, 5, 15 mmol/l) had no effect on tyrosine release from hemi-atria. Five times the normal plasma levels of the branched-chain amino acids leucine, isoleucine and valine had no effect on tyrosine release from either hemi-atria or hemi-diaphragm.

  7. Introduction to protein crystallization

    PubMed Central

    McPherson, Alexander; Gavira, Jose A.

    2014-01-01

    Protein crystallization was discovered by chance about 150 years ago and was developed in the late 19th century as a powerful purification tool and as a demonstration of chemical purity. The crystallization of proteins, nucleic acids and large biological complexes, such as viruses, depends on the creation of a solution that is supersaturated in the macromolecule but exhibits conditions that do not significantly perturb its natural state. Supersaturation is produced through the addition of mild precipitating agents such as neutral salts or polymers, and by the manipulation of various parameters that include temperature, ionic strength and pH. Also important in the crystallization process are factors that can affect the structural state of the macromolecule, such as metal ions, inhibitors, cofactors or other conventional small molecules. A variety of approaches have been developed that combine the spectrum of factors that effect and promote crystallization, and among the most widely used are vapor diffusion, dialysis, batch and liquid–liquid diffusion. Successes in macromolecular crystallization have multiplied rapidly in recent years owing to the advent of practical, easy-to-use screening kits and the application of laboratory robotics. A brief review will be given here of the most popular methods, some guiding principles and an overview of current technologies. PMID:24419610

  8. Peptides and proteins

    SciTech Connect

    Bachovchin, W.W.; Unkefer, C.J.

    1994-12-01

    Advances in magnetic resonance and vibrational spectroscopy make it possible to derive detailed structural information about biomolecular structures in solution. These techniques are critically dependent on the availability of labeled compounds. For example, NMR techniques used today to derive peptide and protein structures require uniformity {sup 13}C-and {sup 15}N-labeled samples that are derived biosynthetically from (U-6-{sup 13}C) glucose. These experiments are possible now because, during the 1970s, the National Stable Isotope Resource developed algal methods for producing (U-6-{sup 13}C) glucose. If NMR techniques are to be used to study larger proteins, we will need sophisticated labelling patterns in amino acids that employ a combination of {sup 2}H, {sup 13}C, and {sup 15}N labeling. The availability of these specifically labeled amino acids requires a renewed investment in new methods for chemical synthesis of labeled amino acids. The development of new magnetic resonance or vibrational techniques to elucidate biomolecular structure will be seriously impeded if we do not see rapid progress in labeling technology. Investment in labeling chemistry is as important as investment in the development of advanced spectroscopic tools.

  9. Cross Talk among TGF-β Signaling Pathways, Integrins, and the Extracellular Matrix

    PubMed Central

    Munger, John S.; Sheppard, Dean

    2011-01-01

    The growth factor TGF-β is secreted in a latent complex consisting of three proteins: TGF-β, an inhibitor (latency-associated protein, LAP, which is derived from the TGF-β propeptide) and an ECM-binding protein (one of the latent TGF-β binding proteins, or LTBPs). LTBPs interact with fibrillins and other ECM components and thus function to localize latent TGF-β in the ECM. LAP contains an integrin-binding site (RGD), and several RGD-binding integrins are able to activate latent TGF-β through binding this site. Mutant mice defective in integrin-mediated activators, and humans and mice with fibrillin gene mutations, show the critical role of ECM and integrins in regulating TGF-β signaling. PMID:21900405

  10. Protein secretion in Pichia pastoris and advances in protein production.

    PubMed

    Damasceno, Leonardo M; Huang, Chung-Jr; Batt, Carl A

    2012-01-01

    Yeast expression systems have been successfully used for over 20 years for the production of recombinant proteins. With the growing interest in recombinant protein expression for various uses, yeast expression systems, such as the popular Pichia pastoris, are becoming increasingly important. Although P. pastoris has been successfully used in the production of many secreted and intracellular recombinant proteins, there is still room for improvement of this expression system. In particular, secretion of recombinant proteins is still one of the main reasons for using P. pastoris. Therefore, endoplasmic reticulum protein folding, correct glycosylation, vesicular transport to the plasma membrane, gene dosage, secretion signal sequences, and secretome studies are important considerations for improved recombinant protein production. PMID:22057543

  11. Neurocognitive derivation of protein surface property from protein aggregate parameters

    PubMed Central

    Mishra, Hrishikesh; Lahiri, Tapobrata

    2011-01-01

    Current work targeted to predicate parametric relationship between aggregate and individual property of a protein. In this approach, we considered individual property of a protein as its Surface Roughness Index (SRI) which was shown to have potential to classify SCOP protein families. The bulk property was however considered as Intensity Level based Multi-fractal Dimension (ILMFD) of ordinary microscopic images of heat denatured protein aggregates which was known to have potential to serve as protein marker. The protocol used multiple ILMFD inputs obtained for a protein to produce a set of mapped outputs as possible SRI candidates. The outputs were further clustered and largest cluster centre after normalization was found to be a close approximation of expected SRI that was calculated from known PDB structure. The outcome showed that faster derivation of individual protein’s surface property might be possible using its bulk form, heat denatured aggregates. PMID:21572883

  12. Side-Chain Conformational Preferences Govern Protein-Protein Interactions.

    PubMed

    Watkins, Andrew M; Bonneau, Richard; Arora, Paramjit S

    2016-08-24

    Protein secondary structures serve as geometrically constrained scaffolds for the display of key interacting residues at protein interfaces. Given the critical role of secondary structures in protein folding and the dependence of folding propensities on backbone dihedrals, secondary structure is expected to influence the identity of residues that are important for complex formation. Counter to this expectation, we find that a narrow set of residues dominates the binding energy in protein-protein complexes independent of backbone conformation. This finding suggests that the binding epitope may instead be substantially influenced by the side-chain conformations adopted. We analyzed side-chain conformational preferences in residues that contribute significantly to binding. This analysis suggests that preferred rotamers contribute directly to specificity in protein complex formation and provides guidelines for peptidomimetic inhibitor design.

  13. Signature Product Code for Predicting Protein-Protein Interactions

    SciTech Connect

    Martin, Shawn B.; Brown, William M.

    2004-09-25

    The SigProdV1.0 software consists of four programs which together allow the prediction of protein-protein interactions using only amino acid sequences and experimental data. The software is based on the use of tensor products of amino acid trimers coupled with classifiers known as support vector machines. Essentially the program looks for amino acid trimer pairs which occur more frequently in protein pairs which are known to interact. These trimer pairs are then used to make predictions about unknown protein pairs. A detailed description of the method can be found in the paper: S. Martin, D. Roe, J.L. Faulon. "Predicting protein-protein interactions using signature products," Bioinformatics, available online from Advance Access, Aug. 19, 2004.

  14. Protein-water dynamics in antifreeze protein III activity

    NASA Astrophysics Data System (ADS)

    Xu, Yao; Bäumer, Alexander; Meister, Konrad; Bischak, Connor G.; DeVries, Arthur L.; Leitner, David M.; Havenith, Martina

    2016-03-01

    We combine Terahertz absorption spectroscopy (THz) and molecular dynamics (MD) simulations to investigate the underlying molecular mechanism for the antifreeze activity of one class of antifreeze protein, antifreeze protein type III (AFP-III) with a focus on the collective water hydrogen bond dynamics near the protein. After summarizing our previous work on AFPs, we present a new investigation of the effects of cosolutes on protein antifreeze activity by adding sodium citrate to the protein solution of AFP-III. Our results reveal that for AFP-III, unlike some other AFPs, the addition of the osmolyte sodium citrate does not affect the hydrogen bond dynamics at the protein surface significantly, as indicated by concentration dependent THz measurements. The present data, in combination with our previous THz measurements and molecular simulations, confirm that while long-range solvent perturbation is a necessary condition for the antifreeze activity of AFP-III, the local binding affinity determines the size of the hysteresis.

  15. Expanding coordination chemistry from protein to protein assembly.

    PubMed

    Sanghamitra, Nusrat J M; Ueno, Takafumi

    2013-05-14

    Bioinorganic chemistry is of growing importance in the fields of nanomaterial science and biotechnology. Coordination of metals by biological systems is a crucial step in intricate enzymatic reactions such as photosynthesis, nitrogen fixation and biomineralization. Although such systems employ protein assemblies as molecular scaffolds, the important roles of protein assemblies in coordination chemistry have not been systematically investigated and characterized. Many researchers are joining the field of bioinorganic chemistry to investigate the inorganic chemistry of protein assemblies. This area is emerging as an important next-generation research field in bioinorganic chemistry. This article reviews recent progress in rational design of protein assemblies in coordination chemistry for integration of catalytic reactions using metal complexes, preparation of mineral biomimetics, and mechanistic investigations of biomineralization processes with protein assemblies. The unique chemical properties of protein assemblies in the form of cages, tubes, and crystals are described in this review.

  16. Signature Product Code for Predicting Protein-Protein Interactions

    2004-09-25

    The SigProdV1.0 software consists of four programs which together allow the prediction of protein-protein interactions using only amino acid sequences and experimental data. The software is based on the use of tensor products of amino acid trimers coupled with classifiers known as support vector machines. Essentially the program looks for amino acid trimer pairs which occur more frequently in protein pairs which are known to interact. These trimer pairs are then used to make predictionsmore » about unknown protein pairs. A detailed description of the method can be found in the paper: S. Martin, D. Roe, J.L. Faulon. "Predicting protein-protein interactions using signature products," Bioinformatics, available online from Advance Access, Aug. 19, 2004.« less

  17. Protein efficiency ratios and net protein ratios of selected protein foods.

    PubMed

    Mitchell, G V; Jenkins, M Y; Grundel, E

    1989-01-01

    As a part of a cooperative study initiated to assess both in vitro and in vivo protein quality methods, the protein efficiency ratio (PER) and net protein ratios (NPR) of 15 different protein sources were determined. Male weanling Sprague-Dawley rats were fed a 10% protein diet. Fourteen-day NPR and relative NPR (RNPR) values and 14- and 28-day PER and relative PER (RPER) values were calculated for each protein source. When protein quality values were expressed relative to ANRC casein, the 14- and 28-day PER data ranked the protein sources essentially in the same order. RPER values of nonfat dried skim milk (unheated) and tuna were more than 100% that of casein; nonfat dried skim milk (heated), chickpeas, and breakfast sausage were between 50 and 70% of that of casein; and pinto beans and rice-wheat gluten cereal did not support substantial growth of the rat. The NPR method did not always rank the protein sources in the same order as the PER method. For the poor quality proteins, RNPR values were much higher than the RPER values; however, the RNPR and RPER values agreed closely for high quality protein sources. PMID:2710752

  18. Proteins interacting with cloning scars: a source of false positive protein-protein interactions.

    PubMed

    Banks, Charles A S; Boanca, Gina; Lee, Zachary T; Florens, Laurence; Washburn, Michael P

    2015-02-23

    A common approach for exploring the interactome, the network of protein-protein interactions in cells, uses a commercially available ORF library to express affinity tagged bait proteins; these can be expressed in cells and endogenous cellular proteins that copurify with the bait can be identified as putative interacting proteins using mass spectrometry. Control experiments can be used to limit false-positive results, but in many cases, there are still a surprising number of prey proteins that appear to copurify specifically with the bait. Here, we have identified one source of false-positive interactions in such studies. We have found that a combination of: 1) the variable sequence of the C-terminus of the bait with 2) a C-terminal valine "cloning scar" present in a commercially available ORF library, can in some cases create a peptide motif that results in the aberrant co-purification of endogenous cellular proteins. Control experiments may not identify false positives resulting from such artificial motifs, as aberrant binding depends on sequences that vary from one bait to another. It is possible that such cryptic protein binding might occur in other systems using affinity tagged proteins; this study highlights the importance of conducting careful follow-up studies where novel protein-protein interactions are suspected.

  19. Prediction of thermodynamic instabilities of protein solutions from simple protein-protein interactions

    NASA Astrophysics Data System (ADS)

    D'Agostino, Tommaso; Solana, José Ramón; Emanuele, Antonio

    2013-10-01

    Statistical thermodynamics of protein solutions is often studied in terms of simple, microscopic models of particles interacting via pairwise potentials. Such modelling can reproduce the short range structure of protein solutions at equilibrium and predict thermodynamics instabilities of these systems. We introduce a square well model of effective protein-protein interaction that embeds the solvent’s action. We modify an existing model [45] by considering a well depth having an explicit dependence on temperature, i.e. an explicit free energy character, thus encompassing the statistically relevant configurations of solvent molecules around proteins. We choose protein solutions exhibiting demixing upon temperature decrease (lysozyme, enthalpy driven) and upon temperature increase (haemoglobin, entropy driven). We obtain satisfactory fits of spinodal curves for both the two proteins without adding any mean field term, thus extending the validity of the original model. Our results underline the solvent role in modulating or stretching the interaction potential.

  20. Geminivirus C3 Protein: Replication Enhancement and Protein Interactions

    PubMed Central

    Settlage, Sharon B.; See, Renee G.; Hanley-Bowdoin, Linda

    2005-01-01

    Most dicot-infecting geminiviruses encode a replication enhancer protein (C3, AL3, or REn) that is required for optimal replication of their small, single-stranded DNA genomes. C3 interacts with C1, the essential viral replication protein that initiates rolling circle replication. C3 also homo-oligomerizes and interacts with at least two host-encoded proteins, proliferating cell nuclear antigen (PCNA) and the retinoblastoma-related protein (pRBR). It has been proposed that protein interactions contribute to C3 function. Using the C3 protein of Tomato yellow leaf curl virus, we examined the impact of mutations to amino acids that are conserved across the C3 protein family on replication enhancement and protein interactions. Surprisingly, many of the mutations did not affect replication enhancement activity of C3 in tobacco protoplasts. Other mutations either enhanced or were detrimental to C3 replication activity. Analysis of mutated proteins in yeast two-hybrid assays indicated that mutations that inactivate C3 replication enhancement activity also reduce or inactivate C3 oligomerization and interaction with C1 and PCNA. In contrast, mutated C3 proteins impaired for pRBR binding are fully functional in replication assays. Hydrophobic residues in the middle of the C3 protein were implicated in C3 interaction with itself, C1, and PCNA, while polar resides at both the N and C termini of the protein are important for C3-pRBR interaction. These experiments established the importance of C3-C3, C3-C1, and C3-PCNA interactions in geminivirus replication. While C3-pRBR interaction is not required for viral replication in cycling cells, it may play a role during infection of differentiated cells in intact plants. PMID:16014949