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Sample records for integrin-binding matricellular protein

  1. 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

  2. Matricellular Proteins in Cardiac Adaptation and Disease

    PubMed Central

    Frangogiannis, Nikolaos G.

    2015-01-01

    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. Thrombospondins (TSP)-1, -2 and -4, 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 pathophysiologic conditions, including myocardial infarction, cardiac hypertrophy and fibrosis, aging-associated myocardial remodeling, myocarditis, diabetic cardiomyopathy and valvular disease. This review manuscript 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. PMID:22535894

  3. Identification of the integrin binding domain of the Yersinia pseudotuberculosis invasin protein.

    PubMed

    Leong, J M; Fournier, R S; Isberg, R R

    1990-06-01

    The invasin protein of the pathogenic Yersinia pseudotuberculosis mediates entry of the bacterium into cultured mammalian cells by binding several beta 1 chain integrins. In this study, we identified the region of invasin responsible for cell recognition. Thirty-two monoclonal antibodies directed against invasin were isolated, and of those, six blocked cell attachment to invasin. These six antibodies recognized epitopes within the last 192 amino acids of invasin. Deletion mutants of invasin and maltose-binding protein (MBP)--invasin fusion proteins were generated and tested for cell attachment. All of the invasin derivatives that carried the carboxyl-terminal 192 amino acids retained cell binding activity. One carboxyl-terminal invasin fragment and seven MBP--invasin fusion proteins were purified. The purified derivatives that retained binding activity inhibited bacterial entry into cultured mammalian cells. These results indicated that the carboxyl-terminal 192 amino acids of invasin contains the integrin-binding domain, even though this region does not contain the tripeptide sequence Arg-Gly-Asp. PMID:1693333

  4. 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. PMID:27135623

  5. 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

  6. 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

  7. 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

  8. 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

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

    PubMed

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

    2015-12-01

    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

  10. 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

  11. 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

  12. 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

  13. The Matricellular Protein CCN1 Promotes Mucosal Healing in Murine Colitis through IL-6

    PubMed Central

    Choi, Jacob S.; Kim, Ki-Hyun; Lau, Lester F.

    2015-01-01

    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 Ccn1dm/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. Ccn1dm/dm mice exhibited high mortality, impaired mucosal healing, and diminished IL-6 expression during the repair phase of DSS-induced colitis compared to 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 Ccn1dm/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. PMID:25807183

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

    PubMed Central

    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. PMID:26890265

  15. The Matricellular Protein CCN1 Suppresses Hepatocarcinogenesis by Inhibiting Compensatory Proliferation

    PubMed Central

    Chen, Chih-Chiun; Kim, Ki-Hyun; Lau, Lester F.

    2015-01-01

    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 down-regulated in human HCC. Ccn1ΔHep mice with hepatocyte-specific deletion of Ccn1 suffer increased HCC tumor multiplicity induced by the hepatocarcinogen diethylnitrosoamine (DEN). Knockin mice (Ccn1dm/dm) that express an integrin α6β1-binding defective CCN1 phenocopied Ccn1ΔHep mice, indicating that CCN1 acts through its α6β1 binding sites in this context. CCN1 effectively inhibits EGFR-dependent hepatocyte proliferation through integrin α6-mediated accumulation of reaction oxygen species (ROS), thereby triggering p53 activation and cell cycle block. Consequently, Ccn1dm/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. PMID:26028023

  16. 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

  17. Integrin binding by Borrelia 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; Coburn, Jenifer

    2015-01-01

    Summary P66, a Borrelia 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

  18. 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.

  19. Cervical Softening During Pregnancy: Regulated Changes in Collagen Cross-Linking and Composition of Matricellular Proteins in the Mouse1

    PubMed Central

    Akins, Meredith L.; Luby-Phelps, Katherine; Bank, Ruud A.; Mahendroo, Mala

    2011-01-01

    A greater understanding of the parturition process is essential in the prevention of preterm birth, which occurs in 12.7% of infants born in the United States annually. Cervical remodeling is a critical component of this process. Beginning early in pregnancy, remodeling requires cumulative, progressive changes in the cervical extracellular matrix (ECM) that result in reorganization of collagen fibril structure with a gradual loss of tensile strength. In the current study, we undertook a detailed biochemical analysis of factors in the cervix that modulate collagen structure during early mouse pregnancy, including expression of proteins involved in processing of procollagen, assembly of collagen fibrils, cross-link formation, and deposition of collagen in the ECM. Changes in these factors correlated with changes in the types of collagen cross-links formed and packing of collagen fibrils as measured by electron microscopy. Early in pregnancy there is a decline in expression of two matricellular proteins, thrombospondin 2 and tenascin C, as well as a decline in expression of lysyl hydroxylase, which is involved in cross-link formation. These changes are accompanied by a decline in both HP and LP cross-links by gestation Days 12 and 14, respectively, as well as a progressive increase in collagen fibril diameter. In contrast, collagen abundance remains constant over the course of pregnancy. We conclude that early changes in tensile strength during cervical softening result in part from changes in the number and type of collagen cross-links and are associated with a decline in expression of two matricellular proteins thrombospondin 2 and tenascin C. PMID:21248285

  20. 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

  1. 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. PMID:26884454

  2. 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

  3. 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

  4. 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

  5. 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

  6. 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

  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. PMID:26933820

  8. 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

  9. Matricellular protein thrombospondin : influence on ocular angiogenesis, wound healing and immuneregulation

    PubMed Central

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

    2014-01-01

    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 (TSP1) and thrombospondin 2 (TSP2) 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 TSP1 and TSP2 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 TSP1. Expressed by many ocular cell types and detectable in the aqueous and vitreous humor TSP1 and TSP2 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 TSP1 and TSP2 in ocular functions and pathology are reviewed here. PMID:24559320

  10. 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

  11. 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. PMID:26698861

  12. 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. PMID:25981593

  13. Adhesive and Migratory Effects of Phosphophoryn Are Modulated by Flanking Peptides of the Integrin Binding Motif

    PubMed Central

    Suzuki, Shigeki; Kobuke, Seiji; Haruyama, Naoto; Hoshino, Hiroaki; Kulkarni, Ashok B.; Nishimura, Fusanori

    2014-01-01

    Phosphophoryn (PP) is generated from the proteolytic cleavage of dentin sialophosphoprotein (DSPP). Gene duplications in the ancestor dentin matrix protein-1 (DMP-1) genomic sequence created the DSPP gene in toothed animals. PP and DMP-1 are phosphorylated extracellular matrix proteins that belong to the family of small integrin-binding ligand N-linked glycoproteins (SIBLINGs). Many SIBLING members have been shown to evoke various cell responses through the integrin-binding Arg-Gly-Asp (RGD) domain; however, the RGD-dependent function of PP is not yet fully understood. We demonstrated that recombinant PP did not exhibit any obvious cell adhesion ability, whereas the simultaneously purified recombinant DMP-1 did. A cell adhesion inhibitory analysis was performed by pre-incubating human osteosarcoma MG63 cells with various PP peptides before seeding onto vitronectin. The results obtained revealed that the incorporation of more than one amino acid on both sides of the PP-RGD domain was unable to inhibit the adhesion of MG63 cells onto vitronectin. Furthermore, the inhibitory activity of a peptide containing the PP-RGD domain with an open carboxyl-terminal side (H-463SDESDTNSESANESGSRGDA482-OH) was more potent than that of a peptide containing the RGD domain with an open amino-terminal side (H-478SRGDASYTSDESSDDDNDSDSH499-OH). This phenomenon was supported by the potent cell adhesion and migration abilities of the recombinant truncated PP, which terminated with Ala482. Furthermore, various point mutations in Ala482 and/or Ser483 converted recombinant PP into cell-adhesive proteins. Therefore, we concluded that the Ala482-Ser483 flanking sequence, which was detected in primates and mice, was the key peptide bond that allowed the PP-RGD domain to be sequestered. The differential abilities of PP and DMP-1 to act on integrin imply that DSPP was duplicated from DMP-1 to serve as a crucial extracellular protein for tooth development rather than as an integrin

  14. 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.

  15. 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

  16. Synthetic integrin-binding peptides promote adhesion and proliferation of human periodontal ligament cells in vitro.

    PubMed

    Grzesik, W J; Ivanov, B; Robey, F A; Southerland, J; Yamauchi, M

    1998-08-01

    Periodontal ligament (PDL) cells have been shown to express several integrins (alphav, alpha5, beta1, beta3) that use RGD (arginine-glycine-aspartic Acid)-dependent mechanisms for the recognition and binding of their ligands. The objective of this study was to evaluate the effects of certain integrin-binding cyclic and linear synthetic RGD-containing peptides on PDL cells' adhesion, proliferation, and de novo protein synthesis in vitro. Fifth passages of normal human PDL cells established from teeth extracted from patients (ages 12 to 14) for orthodontic reasons were used for all experiments. Synthetic peptides containing the EPRGDNYR sequence in two different spatial conformations (linear and cyclic) were covalently attached to bovine serum albumin (BSA). Type I collagen, EPRGDNYR-BSA conjugates, 1:1 mixtures of type I collagen and conjugates, as well as BSA (a negative control) were coated on bacteriological plastic and evaluated for their attachment-promoting activities. In addition, the effects of these substrates on cell proliferation were evaluated by [3H]thymidine incorporation by the PDL cells. For attachment and spreading, the cyclic forms of EPRGDNYR-BSA conjugate and type I collagen were most potent, followed by linear EPRGDNYR-BSA conjugate. The effects of all collagen/conjugate mixtures were equivalent to that of type I collagen except for the collagen/linear EPRGDNYR-BSA mixture, which was less potent. The cyclic EPRGDNYR-BSA conjugate was the most effective substrate to stimulate cell proliferation, and it was followed in potency by the linear peptide-BSA conjugate. Collagen alone did not stimulate [3H]thymidine incorporation above the control level. Mixtures of collagen with all of the conjugates showed stimulatory effects similar to that of the cyclic peptide-BSA conjugate. No significant differences in de novo protein synthesis were detected. These results suggest that the synthetic RGD-containing peptides attached to a carrier are potent ligands

  17. Inclusion of an RGD Motif Alters Invasin Integrin-Binding Affinity and Specificity.

    PubMed

    Khan, Tarik A; Wang, Xianzhe; Maynard, Jennifer A

    2016-04-12

    Invasin is a key adhesin displayed on the outer membrane of Yersinia enterocolitica and Y. pseudotuberculosis that mediates the initial stages of infection. Invasin specifically targets microfold (M) cells in the small intestine by binding β1 integrins and is sufficient to trigger eukaryotic uptake of invasin-coated particles, including Yersinia, Escherichia coli, and latex beads. As a result, invasin has generated interest to mediate oral delivery of vaccines and other biologics. Integrin binding affinity has been shown to correlate with particle uptake; thus we hypothesized that invasin variants with higher affinity would confer enhanced internalization. We first performed alanine scanning of surface-exposed tyrosine residues to identify those contributing to integrin binding. We identified two residues, which, when substituted with alanine, reduced binding to soluble α5β1 integrin. Next, we constructed four targeted mutagenesis libraries spanning these and other residues known to contribute to binding, followed by enrichment of variants able to mediate Caco-2 cellular invasion and to bind soluble α5β1 integrin. We identified three amino acid substitutions that increased α5β1 integrin binding affinity as measured by flow cytometry and ELISA assays, two of which created a novel RGD motif surrounding the D911 residue critical for binding. This variant confers enhanced internalization into CHO cells but not Caco-2 cells when expressed on the E. coli surface. Further analysis showed that inclusion of an RGD expands invasin-integrin specificity, thereby impacting cellular selectivity. This work provides a molecular explanation for the lack of an RGD motif in invasin that is present in many other adhesins. PMID:27015583

  18. 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.

  19. 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. PMID:27089513

  20. Integrin-fibronectin interactions at the cell-material interface: initial integrin binding and signaling.

    PubMed

    García, A J; Boettiger, D

    1999-12-01

    Integrin receptors mediate cell adhesion to extracellular matrices and provide signals that direct proliferation and differentiation. Integrin binding involves receptor-ligand interactions at the cell-substrate interface and assembly and reorganization of structural and signaling elements at the cytoplasmic face. Using a cross-linking/extraction/reversal method to quantify bound integrins, we demonstrate that the density of alpha5beta1 integrin-fibronectin bonds increases linearly with ligand density, as predicted by simple receptor-ligand equilibrium. This linear relationship is consistent with linear increases in cell adhesion strength with receptor and ligand surface densities. Furthermore, we show that phosphorylation of FAK, a tyrosine kinase involved in early integrin-mediated signaling, increases linearly with the number of integrin-Fn bonds. These linear relationships suggest the absence of cooperative effects in the initial stages of mechanical coupling and adhesion-mediated signaling. PMID:10614947

  1. 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

  2. Family with sequence similarity member 20C is the primary but not the only kinase for the small-integrin-binding ligand N-linked glycoproteins in bone.

    PubMed

    Yang, Xiudong; Yan, Wenjuan; Tian, Ye; Ma, Pan; Opperman, Lynne A; Wang, Xiaofang

    2016-01-01

    Recent studies have identified family with sequence similarity member 20C (FAM20C) as a kinase that phosphorylates the Ser in Ser-X-Glu/phospho-Ser (pSer) motifs in the small-integrin-binding ligand N-linked glycoproteins (SIBLINGs). There is no in vivo evidence that validates this finding, and it is unclear whether FAM20C is the only kinase for SIBLINGs. We extracted bone noncollagenous proteins (NCPs) from Fam20C-knockout (KO) mice and analyzed the phosphorylation levels. The total NCPs were separated into osteopontin-, bone sialoprotein-, and dentin matrix protein-1-enriched fractions by anion-exchange chromatography and analyzed by SDS-PAGE, native PAGE, and Western immunoblot analysis. The NCP phosphorylation level in the KO mice was lower than that in the wild-type (WT). On the native gel, the SIBLINGs from KO mice showed a lower migration rate (Mr) than those from the WT. Calf intestine phosphatase treatment shifted SIBLINGs from the WT mice to the level adjacent to the KO, but failed to shift the latter, suggesting a phosphorylation loss of SIBLINGs in the KO mice. Mass spectrometry identified less pSers in the SIBLINGs from the KO mice [including the region of the acidic Ser- and aspartate-rich motif (ASARM) peptides]. In an intriguing finding, several pSers in the Ser-X-Glu motifs in the KO mice maintained their phosphorylation, whereas several others in non-Ser-X-Glu motifs did not. Phospho-Tyrs and phospho-Thrs in the SIBLINGs did not appear to be associated with FAM20C. Our results indicate that FAM20C is the primary, but not the only, kinase for the SIBLINGs.-Yang, X., Yan, W., Tian, Y., Ma, P., Opperman, L. A., Wang, X. Family with sequence similarity member 20C is the primary but not the only kinase for the small-integrin-binding ligand N-linked glycoproteins in bone. PMID:26324849

  3. Crystal structure of the zymogen form of the group A Streptococcus virulence factor SpeB: an integrin-binding cysteine protease.

    PubMed

    Kagawa, T F; Cooney, J C; Baker, H M; McSweeney, S; Liu, M; Gubba, S; Musser, J M; Baker, E N

    2000-02-29

    Pathogenic bacteria secrete protein toxins that weaken or disable their host, and thereby act as virulence factors. We have determined the crystal structure of streptococcal pyrogenic exotoxin B (SpeB), a cysteine protease that is a major virulence factor of the human pathogen Streptococcus pyogenes and participates in invasive disease episodes, including necrotizing fasciitis. The structure, determined for the 40-kDa precursor form of SpeB at 1.6-A resolution, reveals that the protein is a distant homologue of the papain superfamily that includes the mammalian cathepsins B, K, L, and S. Despite negligible sequence identity, the protease portion has the canonical papain fold, albeit with major loop insertions and deletions. The catalytic site differs from most other cysteine proteases in that it lacks the Asn residue of the Cys-His-Asn triad. The prosegment has a unique fold and inactivation mechanism that involves displacement of the catalytically essential His residue by a loop inserted into the active site. The structure also reveals the surface location of an integrin-binding Arg-Gly-Asp (RGD) motif that is a feature unique to SpeB among cysteine proteases and is linked to the pathogenesis of the most invasive strains of S. pyogenes. PMID:10681429

  4. Crystal structure of the zymogen form of the group A Streptococcus virulence factor SpeB: An integrin-binding cysteine protease

    PubMed Central

    Kagawa, Todd F.; Cooney, Jakki C.; Baker, Heather M.; McSweeney, Sean; Liu, Mengyao; Gubba, Siddeswar; Musser, James M.; Baker, Edward N.

    2000-01-01

    Pathogenic bacteria secrete protein toxins that weaken or disable their host, and thereby act as virulence factors. We have determined the crystal structure of streptococcal pyrogenic exotoxin B (SpeB), a cysteine protease that is a major virulence factor of the human pathogen Streptococcus pyogenes and participates in invasive disease episodes, including necrotizing fasciitis. The structure, determined for the 40-kDa precursor form of SpeB at 1.6-Å resolution, reveals that the protein is a distant homologue of the papain superfamily that includes the mammalian cathepsins B, K, L, and S. Despite negligible sequence identity, the protease portion has the canonical papain fold, albeit with major loop insertions and deletions. The catalytic site differs from most other cysteine proteases in that it lacks the Asn residue of the Cys-His-Asn triad. The prosegment has a unique fold and inactivation mechanism that involves displacement of the catalytically essential His residue by a loop inserted into the active site. The structure also reveals the surface location of an integrin-binding Arg-Gly-Asp (RGD) motif that is a feature unique to SpeB among cysteine proteases and is linked to the pathogenesis of the most invasive strains of S. pyogenes. PMID:10681429

  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. Structural insights into how the MIDAS ion stabilizes integrin binding to an RGD peptide under force.

    PubMed

    Craig, David; Gao, Mu; Schulten, Klaus; Vogel, Viola

    2004-11-01

    Integrin alpha(V)beta(3) binds to extracellular matrix proteins through the tripeptide Arg-Gly-Asp (RGD), forming a shallow crevice rather than a deep binding pocket. A dynamic picture of how the RGD-alpha(V)beta(3) complex resists dissociation by mechanical force is derived here from steered molecular dynamic (SMD) simulations in which the major force peak correlates with the breaking of the contact between Asp(RGD) and the MIDAS ion. SMD predicts that the RGD-alpha(V)beta(3) complex is stabilized from dissociation by a single water molecule tightly coordinated to the divalent MIDAS ion, thereby blocking access of free water molecules to the most critical force-bearing interaction. The MIDAS motif is common to many other proteins that contain the phylogenetically ancient von Willebrand A (vWA) domain. The functional role of single water molecules tightly coordinated to the MIDAS ion might reflect a general strategy for the stabilization of protein-protein adhesion against cell-derived forces through divalent cations. PMID:15530369

  7. Selective Detection of RGD-Integrin Binding in Cancer Cells Using Tip Enhanced Raman Scattering Microscopy.

    PubMed

    Xiao, Lifu; Wang, Hao; Schultz, Zachary D

    2016-06-21

    Ligand-receptor interactions play important roles in many biological processes. Cyclic arginine-glycine-aspartic acid (RGD) containing peptides are known to mimic the binding domain of extracellular matrix protein fibronectin and selectively bind to a subset of integrin receptors. Here we report the tip enhanced Raman scattering (TERS) detection of RGD-functionalized nanoparticles bound to integrins produces a Raman scattering signal specific to the bound protein. These results demonstrate that this method can detect and differentiate between two different integrins (α5β1 and αvβ3) bound to RGD-conjugated gold nanoparticles both on surfaces and in a cancer cell membrane. In situ measurements of RGD nanoparticles bound to purified α5β1 and αvβ3 receptors attached to a glass surface provide reference spectra for a multivariate regression model. The TERS spectra observed from nanoparticles bound to cell membranes are analyzed using this regression model and the identity of the receptor can be determined. The ability to distinguish between receptors in the cell membrane provides a new tool to chemically characterize ligand-receptor recognition at molecular level and provide chemical perspective on the molecular recognition of membrane receptors. PMID:27189228

  8. The Adenovirus Type 3 Dodecahedron's RGD Loop Comprises an HSPG Binding Site That Influences Integrin Binding

    PubMed Central

    Gout, E.; Schoehn, G.; Fenel, D.; Lortat-Jacob, H.; Fender, P.

    2010-01-01

    Human type 3 adenovirus dodecahedron (a virus like particle made of twelve penton bases) features the ability to enter cells through Heparan Sulphate Proteoglycans (HSPGs) and integrins interaction and is used as a versatile vector to deliver DNA or proteins. Cryo-EM reconstruction of the pseudoviral particle with Heparan Sulphate (HS) oligosaccharide shows an extradensity on the RGD loop. A set of mutants was designed to study the respective roles of the RGD sequence (RGE mutant) and of a basic sequence located just downstream. Results showed that the RGE mutant binding to the HS deficient CHO-2241 cells was abolished and unexpectedly, mutation of the basic sequence (KQKR to AQAS) dramatically decreased integrin recognition by the viral pseudoparticle. This basic sequence is thus involved in integrin docking, showing a close interplay between HSPGs and integrin receptors. PMID:20224646

  9. LFA-1 and Mac-1 integrins bind to the serine/threonine-rich domain of thrombomodulin.

    PubMed

    Kawamoto, Eiji; Okamoto, Takayuki; Takagi, Yoshimi; Honda, Goichi; Suzuki, Koji; Imai, Hiroshi; Shimaoka, Motomu

    2016-05-13

    LFA-1 (αLβ2) and Mac-1 (αMβ2) integrins regulate leukocyte trafficking in health and disease by binding primarily to IgSF ligand ICAM-1 and ICAM-2 on endothelial cells. Here we have shown that the anti-coagulant molecule thrombomodulin (TM), found on the surface of endothelial cells, functions as a potentially new ligand for leukocyte integrins. We generated a recombinant extracellular domain of human TM and Fc fusion protein (TM-domains 123-Fc), and showed that pheripheral blood mononuclear cells (PBMCs) bind to TM-domains 123-Fc dependent upon integrin activation. We then demonstrated that αL integrin-blocking mAb, αM integrin-blocking mAb, and β2 integrin-blocking mAb inhibited the binding of PBMCs to TM-domains 123-Fc. Furthermore, we show that the serine/threonine-rich domain (domain 3) of TM is required for the interaction with the LFA-1 (αLβ2) and Mac-1 (αMβ2) integrins to occur on PBMCs. These results demonstrate that the LFA-1 and Mac-1 integrins on leukocytes bind to TM, thereby establishing the molecular and structural basis underlying LFA-1 and Mac-1 integrin interaction with TM on endothelial cells. In fact, integrin-TM interactions might be involved in the dynamic regulation of leukocyte adhesion with endothelial cells. PMID:27055590

  10. 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

  11. 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

  12. 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. PMID:26869928

  13. 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

  14. 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

  15. Solution structure of gamma-bungarotoxin: the functional significance of amino acid residues flanking the RGD motif in integrin binding.

    PubMed

    Shiu, Jia-Hau; Chen, Chiu-Yueh; Chang, Long-Sen; Chen, Yi-Chun; Chen, Yen-Chin; Lo, Yu-Hui; Liu, Yu-Chen; Chuang, Woei-Jer

    2004-12-01

    Gamma-bungarotoxin, a snake venom protein isolated from Bungarus multicinctus, contains 68 amino acids, including 10 cysteine residues and a TAVRGDGP sequence at positions 30-37. The solution structure of gamma-bungarotoxin has been determined by nuclear magnetic resonance (NMR) spectroscopy. The structure is similar to that of the short-chain neurotoxins that contain three loops extending from a disulfide-bridged core. The tripeptide Arg-Gly-Asp (RGD) sequence is located at the apex of the flexible loop and is similar to that of other RGD-containing proteins. However, gamma-bungarotoxin only inhibits platelet aggregations with an IC50 of 34 microM. To understand its weak activity in inhibiting platelet aggregation, we mutated the RGD loop sequences of rhodostomin, a potent platelet aggregation inhibitor, from RIPRGDMP to TAVRGDGP, resulting in a 196-fold decrease in activity. In addition, the average Calpha-to-Calpha distance between R33 and G36 of gamma-bungarotoxin is 6.02 A, i.e., shorter than that of other RGD-containing proteins that range from 6.55 to 7.46 A. These results suggested that the amino acid residues flanking the RGD motif might control the width of the RGD loop. This structural difference may be responsible for its decrease in platelet aggregation inhibition compared with other RGD-containing proteins. PMID:15390258

  16. Novel α2β1 Integrin Inhibitors Reveal That Integrin Binding to Collagen under Shear Stress Conditions Does Not Require Receptor Preactivation*

    PubMed Central

    Nissinen, Liisa; Koivunen, Jarkko; Käpylä, Jarmo; Salmela, Maria; Nieminen, Jonna; Jokinen, Johanna; Sipilä, Kalle; Pihlavisto, Marjo; Pentikäinen, Olli T.; Marjamäki, Anne; Heino, Jyrki

    2012-01-01

    The interaction between α2β1 integrin (GPIa/IIa, VLA-2) and vascular collagen is one of the initiating events in thrombus formation. Here, we describe two structurally similar sulfonamide derivatives, BTT-3033 and BTT-3034, and show that, under static conditions, they have an almost identical effect on α2-expressing CHO cell adhesion to collagen I, but only BTT-3033 blocks platelet attachment under flow (90 dynes/cm2). Differential scanning fluorimetry showed that both molecules bind to the α2I domain of the recombinant α2 subunit. To further study integrin binding mechanism(s) of the two sulfonamides, we created an α2 Y285F mutant containing a substitution near the metal ion-dependent adhesion site motif in the α2I domain. The action of BTT-3033, unlike that of BTT-3034, was dependent on Tyr-285. In static conditions BTT-3034, but not BTT-3033, inhibited collagen binding by an α2 variant carrying a conformationally activating E318W mutation. Conversely, in under flow conditions (90 dynes/cm2) BTT-3033, but not BTT-3034, inhibited collagen binding by an α2 variant expressing E336A loss-of-function mutation. Thus, the binding sites for BTT-3033 and BTT-3034 are differentially available in distinct integrin conformations. Therefore, these sulfonamides can be used to study the biological role of different functional stages of α2β1. Furthermore, only the inhibitor that recognized the non-activated conformation of α2β1 integrin under shear stress conditions effectively blocked platelet adhesion, suggesting that the initial interaction between integrin and collagen takes place prior to receptor activation. PMID:23132859

  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. 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

  19. The Matricellular Protein CCN1 Mediates Neutrophil Efferocytosis in Cutaneous Wound Healing

    PubMed Central

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

    2015-01-01

    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 Leprdb/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. PMID:26077348

  20. 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. PMID:26174800

  1. Intrinsically disordered proteins and biomineralization.

    PubMed

    Boskey, Adele L; Villarreal-Ramirez, Eduardo

    2016-01-01

    In vertebrates and invertebrates, biomineralization is controlled by the cell and the proteins they produce. A large number of these proteins are intrinsically disordered, gaining some secondary structure when they interact with their binding partners. These partners include the component ions of the mineral being deposited, the crystals themselves, the template on which the initial crystals form, and other intrinsically disordered proteins and peptides. This review speculates why intrinsically disordered proteins are so important for biomineralization, providing illustrations from the SIBLING (small integrin binding N-glycosylated) proteins and their peptides. It is concluded that the flexible structure, and the ability of the intrinsically disordered proteins to bind to a multitude of surfaces is crucial, but details on the precise-interactions, energetics and kinetics of binding remain to be determined. PMID:26807759

  2. 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

  3. Design of an Osteoinductive Extracellular Fibronectin Matrix Protein for Bone Tissue Engineering

    PubMed Central

    Lee, Sujin; Lee, Dong-Sung; Choi, Ilsan; Pham, Le B. Hang; Jang, Jun-Hyeog

    2015-01-01

    Integrin-mediated cell-matrix interactions play an important role in osteogenesis. Here, we constructed a novel osteoinductive fibronectin matrix protein (oFN) for bone tissue engineering, designed to combine the integrin-binding modules from fibronectin (iFN) and a strong osteoinductive growth factor, bone morphogenetic protein-2. Compared with iFN, the purified oFN matrix protein caused a significant increase in cell adhesion and osteogenic differentiation of pre-osteoblast MC3T3-E1 cells (p < 0.05). PMID:25853265

  4. 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

  5. Stromal Fibrosis and Expression of Matricellular Proteins Correlate With Histological Grade of Intraductal Papillary Mucinous Neoplasm of the Pancreas

    PubMed Central

    Kakizaki, Yasuharu; Makino, Naohiko; Tozawa, Tomohiro; Honda, Teiichiro; Matsuda, Akiko; Ikeda, Yushi; Ito, Miho; Saito, Yoshihiko; Kimura, Wataru; Ueno, Yoshiyuki

    2016-01-01

    Objective The aim of the study was to clarify the correlation between the microenvironmental factors and histological grade in intraductal papillary mucinous neoplasm (IPMN). Methods We investigated 65 IPMNs resected at Yamagata University Hospital between 2000 and 2011, and all cases were categorized to low-inter (including low- and intermediate-grade dysplasia) and high-inv (including high-grade dysplasia and IPMN with an associated invasive carcinoma) groups. We compared between the 2 groups pathologically with regard to fibrosis and the expression of alpha-smooth muscle actin (α-SMA), periostin, and galectin-1 in the periductal stroma of IPMN. Results There were 41 low-inter and 24 high-inv. The subtype was categorized as 22 main duct type (MD-IPMN) and 43 branch duct type (BD-IPMN). The degree of fibrosis and the expression of α-SMA, periostin, and galectin-1 were significantly higher in high-inv than in low-inter within BD-IPMNs. Multivariate logistic regression analysis indicated that high expression of α-SMA (odds ratio, 13.802; 95% confidence interval, 1.108–171.893; P = 0.0414) was a significant independent related factor of high-inv in BD-IPMN. Conclusions Stromal fibrosis and expression of α-SMA, periostin, and galectin-1 are more marked in high-inv than in low-inter within BD-IPMNs, and they could become new markers for determining the indications for surgery in BD-IPMN. PMID:26967452

  6. Engineering multiple biological functional motifs into a blank collagen-like protein template from Streptococcus pyogenes.

    PubMed

    Peng, Yong Y; Stoichevska, Violet; Schacht, Kristin; Werkmeister, Jerome A; Ramshaw, John A M

    2014-07-01

    Bacterially derived triple-helical, collagen-like proteins are attractive as potential biomedical materials. The collagen-like domain of the Scl2 protein from S. pyogenes lacks any specific binding sites for mammalian cells yet possesses the inherent structural integrity of the collagen triple-helix of animal collagens. It can, therefore, be considered as a structurally-stable "blank slate" into which various defined, biological sequences, derived from animal collagens, can be added by substitutions or insertions, to enable production of novel designed materials to fit specific functional requirements. In the present study, we have used site directed mutagenesis to substitute two functional sequences, one for heparin binding and the other for integrin binding, into different locations in the triple-helical structure. This provided three new constructs, two containing the single substitutions and one containing both substitutions. The stability of these constructs was marginally reduced when compared to the unmodified sequence. When compared to the unmodified bacterial collagen, both the modified collagens that contain the heparin binding site showed marked binding of fluorescently labeled heparin. Similarly, the modified collagens from both constructs containing the integrin binding site showed significant adhesion of L929 cells that are known to possess the appropriate integrin receptor. C2C12 cells that lack any appropriate integrins did not bind. These data show that bacterial collagen-like sequences can be modified to act like natural extracellular matrix collagens by inserting one or more unique biological domains with defined function. PMID:23913780

  7. 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.

  8. The role of secreted protein acidic and rich in cysteine (SPARC) in cardiac repair and fibrosis: Does expression of SPARC by macrophages influence outcomes?

    PubMed

    Bradshaw, Amy D

    2016-04-01

    Secreted protein acidic and rich in cysteine (SPARC) is a matricellular, collagen-binding protein. Matricellular proteins are described as extracellular matrix-associated proteins that do not serve classical structural roles in the matrix such as those ascribed to laminins and collagens. The family of matricellular proteins modulates cell:extracellular matrix interactions and is actively expressed during tissue remodeling. Functional activities attributed to SPARC in cultured cells include regulation of cell adhesion, cytoskeletal rearrangement, proliferation, and matrix assembly. The primary phenotype characteristic of SPARC-null mice is a deficit in amounts of fibrillar collagen and fibril morphology. Strikingly, SPARC-null mice demonstrate a blunted fibrotic response in a number of different tissue settings. The role of monocyte/macrophages as an important component of tissue fibrosis is becoming increasingly appreciated. Expression of SPARC by bone marrow derived inflammatory cells raises the interesting proposition that SPARC produced by infiltrating leukocytes might contribute to the course of inflammation and tissue fibrosis in the heart. This review will summarize results from studies defining the function of SPARC in myocardial repair and fibrosis and results from other non-cardiac tissues that shed light onto possible consequences of SPARC expression by monocyte/macrophages in the setting of heart disease. PMID:26582465

  9. 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. PMID:27098435

  10. 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

  11. The Human Metapneumovirus Fusion Protein Mediates Entry via an Interaction with RGD-Binding Integrins

    PubMed Central

    Cox, Reagan G.; Livesay, S. Brent; Johnson, Monika; Ohi, Melanie D.

    2012-01-01

    Paramyxoviruses use a specialized fusion protein to merge the viral envelope with cell membranes and initiate infection. Most paramyxoviruses require the interaction of two viral proteins to enter cells; an attachment protein binds cell surface receptors, leading to the activation of a fusion (F) protein that fuses the viral envelope and host cell plasma membrane. In contrast, human metapneumovirus (HMPV) expressing only the F protein is replication competent, suggesting a primary role for HMPV F in attachment and fusion. We previously identified an invariant arginine-glycine-aspartate (RGD) motif in the HMPV F protein and showed that the RGD-binding integrin αVβ1-promoted HMPV infection. Here we show that both HMPV F-mediated binding and virus entry depend upon multiple RGD-binding integrins and that HMPV F can mediate binding and fusion in the absence of the viral attachment (G) protein. The invariant F-RGD motif is critical for infection, as an F-RAE virus was profoundly impaired. Further, F-integrin binding is required for productive viral RNA transcription, indicating that RGD-binding integrins serve as receptors for the HMPV fusion protein. Thus, HMPV F is triggered to induce virus-cell fusion by interactions with cellular receptors in a manner that is independent of the viral G protein. These results suggest a stepwise mechanism of HMPV entry mediated by the F protein through its interactions with cellular receptors, including RGD-binding integrins. PMID:22933271

  12. 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

  13. BB0172, a Borrelia burgdorferi Outer Membrane Protein That Binds Integrin α3β1

    PubMed Central

    Wood, Elaine; Tamborero, Silvia; Mingarro, Ismael

    2013-01-01

    Lyme disease is a multisystemic disorder caused by Borrelia burgdorferi infection. Upon infection, some B. burgdorferi genes are upregulated, including members of the microbial surface components recognizing adhesive matrix molecule (MSCRAMM) protein family, which facilitate B. burgdorferi adherence to extracellular matrix components of the host. Comparative genome analysis has revealed a new family of B. burgdorferi proteins containing the von Willebrand factor A (vWFA) domain. In the present study, we characterized the expression and membrane association of the vWFA domain-containing protein BB0172 by using in vitro transcription/translation systems in the presence of microsomal membranes and with detergent phase separation assays. Our results showed evidence of BB0172 localization in the outer membrane, the orientation of the vWFA domain to the extracellular environment, and its function as a metal ion-dependent integrin-binding protein. This is the first report of a borrelial adhesin with a metal ion-dependent adhesion site (MIDAS) motif that is similar to those observed in eukaryotic integrins and has a similar function. PMID:23687274

  14. BB0172, a Borrelia burgdorferi outer membrane protein that binds integrin α3β1.

    PubMed

    Wood, Elaine; Tamborero, Silvia; Mingarro, Ismael; Esteve-Gassent, Maria D

    2013-08-01

    Lyme disease is a multisystemic disorder caused by Borrelia burgdorferi infection. Upon infection, some B. burgdorferi genes are upregulated, including members of the microbial surface components recognizing adhesive matrix molecule (MSCRAMM) protein family, which facilitate B. burgdorferi adherence to extracellular matrix components of the host. Comparative genome analysis has revealed a new family of B. burgdorferi proteins containing the von Willebrand factor A (vWFA) domain. In the present study, we characterized the expression and membrane association of the vWFA domain-containing protein BB0172 by using in vitro transcription/translation systems in the presence of microsomal membranes and with detergent phase separation assays. Our results showed evidence of BB0172 localization in the outer membrane, the orientation of the vWFA domain to the extracellular environment, and its function as a metal ion-dependent integrin-binding protein. This is the first report of a borrelial adhesin with a metal ion-dependent adhesion site (MIDAS) motif that is similar to those observed in eukaryotic integrins and has a similar function. PMID:23687274

  15. Wiskott–Aldrich syndrome protein is involved in αIIbβ3-mediated cell adhesion

    PubMed Central

    Tsuboi, Shigeru; Nonoyama, Shigeaki; Ochs, Hans D

    2006-01-01

    The Wiskott–Aldrich syndrome (WAS) is an X-chromosome-linked immunodeficiency disorder. The most common symptom seen in WAS patients is bleeding. One of the main causes of bleeding is defective platelet aggregation. The causative gene of WAS encodes WAS protein (WASP). Here, we show that WASP binds to the calcium- and integrin-binding protein (CIB) in platelets. CIB was originally identified as a protein binding to the αIIb cytoplasmic tail of platelet integrin αIIbβ3, which has a primary role in platelet aggregation. We also show that the WASP–CIB complex is important in αIIbβ3-mediated cell adhesion, and that in patients mutant forms of WASP are expressed at reduced levels or show lower affinities for CIB than wild-type WASP. Our results indicate that impaired complex formation between mutant WASPs and CIB reduces αIIbβ3-mediated cell adhesion and causes defective platelet aggregation, resulting in bleeding. PMID:16582881

  16. 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...

  17. 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)

  18. 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.

  19. A Short-Term Borrelia burgdorferi Infection Model Identifies Tissue Tropisms and Bloodstream Survival Conferred by Adhesion Proteins

    PubMed Central

    Caine, Jennifer A.

    2015-01-01

    Borrelia burgdorferi, the causative agent of Lyme disease in the United States, is able to persist in the joint, heart, skin, and central nervous system for the lifetime of its mammalian host. Borrelia species achieve dissemination to distal sites in part by entry into and travel within the bloodstream. Much work has been performed in vitro describing the roles of many B. burgdorferi outer surface proteins in adhesion to host cell surface proteins and extracellular matrix components, although the biological relevance of these interactions is only beginning to be explored in vivo. A need exists in the field for an in vivo model to define the biological roles of B. burgdorferi adhesins in tissue-specific vascular interactions. We have developed an in vivo model of vascular interaction of B. burgdorferi in which the bacteria are injected intravenously and allowed to circulate for 1 h. This model has shown that the fibronectin binding protein BB0347 has a tropism for joint tissue. We also have shown an importance of the integrin binding protein, P66, in binding to vasculature of the ear and heart. This model also revealed unexpected roles for Borrelia adhesins BBK32 and OspC in bacterial burdens in the bloodstream. The intravenous inoculation model of short-term infection provides new insights into critical B. burgdorferi interactions with the host required for initial survival and tissue colonization. PMID:26015482

  20. 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

  1. 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

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

    PubMed Central

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

    2009-01-01

    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

  3. 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

  4. 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

  5. Molecular cloning and characterisation of a pattern recognition molecule, lipopolysaccharide- and beta-1,3-glucan binding protein (LGBP) from the white shrimp Litopenaeus vannamei.

    PubMed

    Cheng, Winton; Liu, Chun-Hung; Tsai, Chiung-Hui; Chen, Jiann-Chu

    2005-04-01

    A lipopolysaccharide- and beta-1,3-glucan binding protein (LGBP) cDNA was cloned from the haemocyte and hepatopancreas of white shrimp Litopenaeus vannamei using oligonucleotide primers and RT-PCR. Both 3'- and 5'-regions were isolated by rapid amplification of cDNA end RACE method. Analysis of nucleotide sequence revealed that the cDNA clone has an open reading frame of 1101 bp encoding a protein of 367 amino acids including a 17 amino acid signal peptide. The calculated molecular mass of the mature proteins (350 amino acids) is 39.92 kDa with an estimated pI of 4.37. Two putative integrin binding motifs (cell adhesion site), RGD (Arg-Gly-Asp) and a potential recognition motif for beta- (1-->3) linkage of polysaccharides were observed in the LGBP. Sequence comparison showed that LGBP deduced amino acid of L. vannamei has an overall similarity of 95%, 92% and 61% to that of blue shrimp Litopenaeus stylirostris LGBP, tiger shrimp Penaeus monodon BGBP and crayfish Pacifastacus leniusculus LGBP, respectively. Quantitative real-time RT-PCR analysis showed that LGBP transcript in haemocyte of L. vannamei increased in 3- and 6-h post Vibrio alginolyticus injection. PMID:15561560

  6. Fibulin-5 Blocks Microenvironmental ROS in Pancreatic Cancer.

    PubMed

    Wang, Miao; Topalovski, Mary; Toombs, Jason E; Wright, Christopher M; Moore, Zachary R; Boothman, David A; Yanagisawa, Hiromi; Wang, Huamin; Witkiewicz, Agnieszka; Castrillon, Diego H; Brekken, Rolf A

    2015-12-01

    Elevated oxidative stress is an aberration seen in many solid tumors, and exploiting this biochemical difference has the potential to enhance the efficacy of anticancer agents. Homeostasis of reactive oxygen species (ROS) is important for normal cell function, but excessive production of ROS can result in cellular toxicity, and therefore ROS levels must be balanced finely. Here, we highlight the relationship between the extracellular matrix and ROS production by reporting a novel function of the matricellular protein Fibulin-5 (Fbln5). We used genetically engineered mouse models of pancreatic ductal adenocarcinoma (PDAC) and found that mutation of the integrin-binding domain of Fbln5 led to decreased tumor growth, increased survival, and enhanced chemoresponse to standard PDAC therapies. Through mechanistic investigations, we found that improved survival was due to increased levels of oxidative stress in Fbln5-mutant tumors. Furthermore, loss of the Fbln5-integrin interaction augmented fibronectin signaling, driving integrin-induced ROS production in a 5-lipooxygenase-dependent manner. These data indicate that Fbln5 promotes PDAC progression by functioning as a molecular rheostat that modulates cell-ECM interactions to reduce ROS production, and thus tip the balance in favor of tumor cell survival and treatment-refractory disease. PMID:26577699

  7. Differential effects of insulin-like growth factor I (IGF-I) and IGF-binding protein-1 on protein metabolism in human skeletal muscle cells.

    PubMed

    Frost, R A; Lang, C H

    1999-09-01

    Insulin-like growth factor-binding protein-1 (BP-1) is a multifunctional protein that binds IGF-I in solution and integrins on the cell surface. BP-1 is overexpressed during catabolic illnesses, and the protein accumulates in skeletal muscle. To define a potential physiological role for BP-1 in regulating muscle protein balance, we have examined the effect of IGF-I and BP-1 on protein synthesis and degradation in human skeletal muscle cells. IGF-I-stimulated protein synthesis by 20%, and this was completely inhibited by either phosphorylated or nonphosphorylated BP-1. Half-maximal inhibition of protein synthesis occurred at a molar ratio of BP-1 to IGF-I of 1.5:1. BP-1 failed to form a complex with a truncated form of IGF-I (desIGF-I), and consequently, BP-1 failed to inhibit the ability of desIGF-I to stimulate protein synthesis. IGF-I and BP-1 dose-dependently inhibited protein degradation individually, and both BP-1 phosphovariants failed to block the ability of IGF-I to do the same. Blocking integrin receptor occupancy with the integrin antagonist echistatin blunted the ability of BP-1 to inhibit protein degradation, but had no significant effect on IGF-I-mediated changes in protein synthesis or degradation. The extracellular matrix protein vitronectin also inhibited protein degradation, but vitronectin receptor antibodies failed to block BP-1 action. In contrast, antibodies to the beta1 integrin subunit blocked BP-1-mediated inhibition of protein degradation. Rapamycin inhibited IGF-I-dependent protein synthesis, but not the ability of IGF-I to inhibit proteolysis. In contrast, rapamycin completely blocked the ability of BP-1 to inhibit proteolysis. Our results demonstrate that BP-1 inhibits IGF-I-mediated protein synthesis by binding to IGF-I. BP-1, acting independently of IGF-I, inhibits protein degradation. The IGF-independent response occurs via beta1 integrin binding and stimulation of a rapamycin-sensitive signal transduction pathway. PMID:10465265

  8. Identification of a fibronectin interaction site in the extracellular matrix protein ameloblastin.

    PubMed

    Beyeler, Michael; Schild, Christof; Lutz, Roman; Chiquet, Matthias; Trueb, Beat

    2010-04-15

    Mammalian teeth are composed of hydroxyapatite crystals that are embedded in a rich extracellular matrix. This matrix is produced by only two cell types, the mesenchymal odontoblasts and the ectodermal ameloblasts. Ameloblasts secrete the enamel proteins amelogenin, ameloblastin, enamelin and amelotin. Odontoblasts secrete collagen type I and several calcium-binding phosphoproteins including dentin sialophosphoprotein, dentin matrix protein, bone sialoprotein and osteopontin. The latter four proteins have recently been grouped in the family of the SIBLINGs (small integrin-binding ligand, N-linked glycoproteins) because they display similar gene structures and because they contain an RGD tripeptide sequence that binds to integrin receptors and thus mediates cell adhesion. We have prepared all the other tooth-specific proteins in recombinant form and examined whether they might also promote cell adhesion similar to the SIBLINGs. We found that only ameloblastin consistently mediated adhesion of osteoblastic and fibroblastic cells to plastic or titanium surfaces. The activity was dependent on the intact three-dimensional structure of ameloblastin and required de novo protein synthesis of the adhering cells. By deletion analysis and in vitro mutagenesis, the active site could be narrowed down to a sequence of 13 amino acid residues (VPIMDFADPQFPT) derived from exon 7 of the rat ameloblastin gene or exons 7-9 of the human gene. Kinetic studies and RNA interference experiments further demonstrated that this sequence does not directly bind to a cell surface receptor but that it interacts with cellular fibronectin, which in turn binds to integrin receptors. The identification of a fibronectin-binding domain in ameloblastin might permit interesting applications for dental implantology. Implants could be coated with peptides containing the active sequence, which in turn would recruit fibronectin from the patient's blood. The recruited fibronectin should then promote cell

  9. Bone morphogenetic protein 7 induces cementogenic differentiation of human periodontal ligament-derived mesenchymal stem cells.

    PubMed

    Torii, D; Tsutsui, T W; Watanabe, N; Konishi, K

    2016-01-01

    Bone morphogenetic protein 7 (BMP-7) is a multifunctional differentiation factor that belongs to the transforming growth factor superfamily. BMP-7 induces gene expression of protein tyrosine phosphatase-like, member A/cementum attachment protein (PTPLA/CAP) and cementum protein 1 (CEMP1), both of which are markers of cementoblasts and cementocytes. In the previous study, we reported that BMP-7 treatment enhanced PTPLA/CAP and CEMP1 expression in both normal and immortal human periodontal ligament (PDL) cells. To elucidate the molecular mechanisms of the gene expression of these molecules, in this study, we identified a functional transcription activator binding region in the promoter region of PTPLA/CAP and CEMP1 that is responsive to BMP signals. Here, we report that some short motifs termed GC-rich Smad-binding elements (GC-SBEs) that are located in the human PTPLA/CAP promoter and CEMP1 promoter are BMP-7 responsive as analyzed with luciferase promoter assays. On the other hand, we found that transcription of Sp7/Osterix and PTPLA/CAP was up-regulated after 1 week of BMP-7 treatment on purified normal human PDL cells as a result of gene expression microarray analysis. Furthermore, transcription of Sp7/Osterix, runt-related transcription factor 2 (RUNX2), and alkaline phosphatase (ALP) was up-regulated after 2 weeks of BMP-7 treatment, whereas gene expression of osteo/odontogenic markers such as integrin-binding sialoprotein (IBSP), collagen, type I, alpha 1 (COL1A1), dentin matrix acidic phosphoprotein 1 (DMP1), and dentin sialophosphoprotein (DSPP) was not up-regulated in purified normal or immortal human PDL cells as a result of qRT-PCR. The results suggest that BMP-7 mediates cementogenesis via GC-SBEs in human PDL cells and that its molecular mechanism is different from that for osteo/odontogenesis. PMID:25464857

  10. In vivo adhesion of malignant B cells to bone marrow microvasculature is regulated by α4β1 cytoplasmic-binding proteins.

    PubMed

    Martínez-Moreno, M; Leiva, M; Aguilera-Montilla, N; Sevilla-Movilla, S; Isern de Val, S; Arellano-Sánchez, N; Gutiérrez, N C; Maldonado, R; Martínez-López, J; Buño, I; García-Marco, J A; Sánchez-Mateos, P; Hidalgo, A; García-Pardo, A; Teixidó, J

    2016-04-01

    Multiple myeloma (MM) and chronic lymphocytic leukemia (CLL) cells must attach to the bone marrow (BM) microvasculature before lodging in the BM microenvironment. Using intravital microscopy (IVM) of the BM calvariae we demonstrate that the α4β1 integrin is required for MM and CLL cell firm arrest onto the BM microvasculature, while endothelial P-selectin and E-selectin mediate cell rolling. Talin, kindlin-3 and ICAP-1 are β1-integrin-binding partners that regulate β1-mediated cell adhesion. We show that talin and kindlin-3 cooperatively stimulate high affinity and strength of α4β1-dependent MM and CLL cell attachment, whereas ICAP-1 negatively regulates this adhesion. A functional connection between talin/kindlin-3 and Rac1 was found to be required for MM cell attachment mediated by α4β1. Importantly, IVM analyses with talin- and kindlin-3-silenced MM cells indicate that these proteins are needed for cell arrest on the BM microvasculature. Instead, MM cell arrest is repressed by ICAP-1. Moreover, MM cells silenced for talin and kindlin-3, and cultured on α4β1 ligands showed higher susceptibility to bortezomib-mediated cell apoptosis. Our results highlight the requirement of α4β1 and selectins for the in vivo attachment of MM and CLL cells to the BM microvasculature, and indicate that talin, kindlin-3 and ICAP-1 differentially control physiological adhesion by regulating α4β1 activity. PMID:26658839

  11. 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

  12. 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.

  13. Ligand-specific, transient interaction between integrins and calreticulin during cell adhesion to extracellular matrix proteins is dependent upon phosphorylation/dephosphorylation events.

    PubMed Central

    Coppolino, M G; Dedhar, S

    1999-01-01

    As transmembrane heterodimers, integrins bind to both extracellular ligands and intracellular proteins. We are currently investigating the interaction between integrins and the intracellular protein calreticulin. A prostatic carcinoma cell line (PC-3) was used to demonstrate that calreticulin can be found in the alpha3 immunoprecipitates of cells plated on collagen type IV, but not when plated on vitronectin. Conversely, alphav immunoprecipitates contained calreticulin only when cells were plated on vitronectin, i. e. not when plated on collagen IV. The interactions between these integrins and calreticulin were independent of actin cytoskeleton assembly and were transient, being maximal approx. 10-30 min after the cells came into contact with the substrates prior to complete cell spreading and formation of firm adhesive contacts. We demonstrate that okadaic acid, an inhibitor of intracellular serine/threonine protein phosphatases, inhibited the alpha3beta1-mediated adhesion of PC-3 cells to collagen IV and the alpha2beta1-mediated attachment of Jurkat cells to collagen I. This inhibition by okadaic acid was accompanied by inhibition of the ligand-specific interaction of calreticulin with the respective integrins in the two cell types. Additionally, we found that pharmacological inhibition of mitogen-activated protein kinase kinase (MEK) resulted in prolongation of the calreticulin-integrin interaction, and enhancement of PC-3 cell attachment to collagen IV. We conclude that calreticulin interacts transiently with integrins during cell attachment and spreading. This interaction depends on receptor occupation, is ligand-specific, and can be modulated by protein phosphatase and MEK activity. PMID:10229657

  14. 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.

  15. 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.

  16. 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

  17. 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

  18. The structure of the Ca{sup 2+}-binding , glycosylated F-spondin domain of F-spondin- A C2-domain variant in an extracellular matrix protein.

    SciTech Connect

    Tan, K.; Lawler, J.

    2011-05-10

    F-spondin is a multi-domain extracellular matrix (ECM) protein and a contact-repellent molecule that directs axon outgrowth and cell migration during development. The reelin{_}N domain and the F-spondin domain (FS domain) comprise a proteolytic fragment that interacts with the cell membrane and guides the projection of commissural axons to floor plate. The FS domain is found in F-spondins, mindins, M-spondin and amphiF-spondin. We present the crystal structure of human F-spondin FS domain at 1.95{angstrom} resolution. The structure reveals a Ca{sup 2+}-binding C2 domain variant with an 8-stranded antiparallel {beta}-sandwich fold. Though the primary sequences of the FS domains of F-spondin and mindin are less than 36% identical, their overall structures are very similar. The unique feature of F-spondin FS domain is the presence of three disulfide bonds associated with the N- and C-termini of the domain and a highly conserved N-linked glycosylation site. The integrin-binding motif found in mindin is not conserved in the F-spondin FS domain. The structure of the F-spondin FS domain completes the structural studies of the multiple-domain ECM molecule. The homology of its core structure to a common Ca{sup 2+}- and lipid-binding C2 domain suggests that the F-spondin FS domain may be responsible for part of the membrane targeting of F-spondin in its regulation of axon development. The structural properties of the FS domain revealed in this study pave the way for further exploration into the functions of F-spondin.

  19. 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.

  20. Plasmodium falciparum Erythrocyte Membrane Protein 1 Diversity in Seven Genomes – Divide and Conquer

    PubMed Central

    Rask, Thomas S.; Hansen, Daniel A.; Theander, Thor G.; Gorm Pedersen, Anders; Lavstsen, Thomas

    2010-01-01

    The var gene encoded hyper-variable Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) family mediates cytoadhesion of infected erythrocytes to human endothelium. Antibodies blocking cytoadhesion are important mediators of malaria immunity acquired by endemic populations. The development of a PfEMP1 based vaccine mimicking natural acquired immunity depends on a thorough understanding of the evolved PfEMP1 diversity, balancing antigenic variation against conserved receptor binding affinities. This study redefines and reclassifies the domains of PfEMP1 from seven genomes. Analysis of domains in 399 different PfEMP1 sequences allowed identification of several novel domain classes, and a high degree of PfEMP1 domain compositional order, including conserved domain cassettes not always associated with the established group A–E division of PfEMP1. A novel iterative homology block (HB) detection method was applied, allowing identification of 628 conserved minimal PfEMP1 building blocks, describing on average 83% of a PfEMP1 sequence. Using the HBs, similarities between domain classes were determined, and Duffy binding-like (DBL) domain subclasses were found in many cases to be hybrids of major domain classes. Related to this, a recombination hotspot was uncovered between DBL subdomains S2 and S3. The VarDom server is introduced, from which information on domain classes and homology blocks can be retrieved, and new sequences can be classified. Several conserved sequence elements were found, including: (1) residues conserved in all DBL domains predicted to interact and hold together the three DBL subdomains, (2) potential integrin binding sites in DBLα domains, (3) an acylation motif conserved in group A var genes suggesting N-terminal N-myristoylation, (4) PfEMP1 inter-domain regions proposed to be elastic disordered structures, and (5) several conserved predicted phosphorylation sites. Ideally, this comprehensive categorization of PfEMP1 will provide a

  1. 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

  2. 111In- and 203Pb-Labeled Cyclic RGD Peptide Conjugate as an αvβ3 Integrin-Binding Radiotracer

    PubMed Central

    Nwe, Kido; Kim, Young-Seung; Milenic, Diane E.; Baidoo, Kwamena E.; Brechbiel, Martin W.

    2012-01-01

    Methodology for site-specific modification and chelate conjugation of a cyclic RGD (cRGD) peptide for the preparation of a radiotracer molecular imaging agent suitable for detecting αvβ3 integrin is described. The method involves functionalizing the peptide with an aldehyde moiety and conjugation to a 1,4,7,10-tetraazacyclododecane-N,N′,N″,N‴-tetraacetic acid (DOTA) derivative that possesses an aldehyde reactive aminooxy group. The binding assay of the 111In-labeled peptide conjugate with αvβ3 integrin showed 60% bound when four equivalents of the integrin was added, a reasonable binding affinity for a mono-valent modified RGD peptide. PMID:23162207

  3. 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.

  4. 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.

  5. Total protein

    MedlinePlus

    The total protein test measures the total amount of two classes of proteins found in the fluid portion of your ... nutritional problems, kidney disease or liver disease . If total protein is abnormal, you will need to have more ...

  6. Storage Proteins

    PubMed Central

    Fujiwara, Toru; Nambara, Eiji; Yamagishi, Kazutoshi; Goto, Derek B.; Naito, Satoshi

    2002-01-01

    Plants accumulate storage substances such as starch, lipids and proteins in certain phases of development. Storage proteins accumulate in both vegetative and reproductive tissues and serve as a reservoir to be used in later stages of plant development. The accumulation of storage protein is thus beneficial for the survival of plants. Storage proteins are also an important source of dietary plant proteins. Here, we summarize the genome organization and regulation of gene expression of storage protein genes in Arabidopsis. PMID:22303197

  7. Dietary Proteins

    MedlinePlus

    ... 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 of plant proteins every day to get ...

  8. Protein Analysis

    NASA Astrophysics Data System (ADS)

    Chang, Sam K. C.

    Proteins are an abundant component in all cells, and almost all except storage proteins are important for biological functions and cell structure. Food proteins are very complex. Many have been purified and characterized. Proteins vary in molecular mass, ranging from approximately 5000 to more than a million Daltons. They are composed of elements including hydrogen, carbon, nitrogen, oxygen, and sulfur. Twenty α-amino acids are the building blocks of proteins; the amino acid residues in a protein are linked by peptide bonds. Nitrogen is the most distinguishing element present in proteins. However, nitrogen content in various food proteins ranges from 13.4 to 19.1% (1) due to the variation in the specific amino acid composition of proteins. Generally, proteins rich in basic amino acids contain more nitrogen.

  9. 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

  10. 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 ...

  11. Whey Protein

    MedlinePlus

    ... shows that taking whey protein in combination with strength training increases lean body mass, strength, and muscle size. ... grams/kg of whey protein in combination with strength training for 6-10 weeks. For HIV/AIDS-related ...

  12. 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.

  13. 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

  14. 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…

  15. Protein folds and protein folding

    PubMed Central

    Schaeffer, R. Dustin; Daggett, Valerie

    2011-01-01

    The classification of protein folds is necessarily based on the structural elements that distinguish domains. Classification of protein domains consists of two problems: the partition of structures into domains and the classification of domains into sets of similar structures (or folds). Although similar topologies may arise by convergent evolution, the similarity of their respective folding pathways is unknown. The discovery and the characterization of the majority of protein folds will be followed by a similar enumeration of available protein folding pathways. Consequently, understanding the intricacies of structural domains is necessary to understanding their collective folding pathways. We review the current state of the art in the field of protein domain classification and discuss methods for the systematic and comprehensive study of protein folding across protein fold space via atomistic molecular dynamics simulation. Finally, we discuss our large-scale Dynameomics project, which includes simulations of representatives of all autonomous protein folds. PMID:21051320

  16. Protein Dynamics

    NASA Astrophysics Data System (ADS)

    Frauenfelder, Hans

    2011-03-01

    Proteins combine properties of solids, liquids, and glasses. Schrödinger anticipated the main features of biomolecules long ago by stating that they had to be solid-like, but able to assume many different conformations. Indeed proteins can assume a gigantic number of conformational substates with the same primary sequence but different conformations. The different substates are described as craters in a very-high-dimensional energy landscape. The energy landscape is organized in a hierarchy of tiers, craters within craters within craters. Protein motions are pictured as transition between substates - jumps from crater to crater. Initially we assumed that these jumps were controlled by internal barriers between substates, but experiments have shown that nature selected a different approach. Proteins are surrounded by one to two layers of water and are embedded in a bulk solvent. Structural motions of the protein are controlled by the alpha fluctuations in the solvent surrounding the protein. Some internal motions most likely involving side chains are controlled electrostatically by beta fluctuations in the hydration shell. The dynamics of proteins is consequently dominated by the environment (H. Frauenfelder et al. PNAS 106, 5129 (2009). One can speculate that this organization permits exchange of information among biomolecules. The energy landscape is not just organized into two tiers, alpha and beta, but cryogenic experiments have revealed more tiers and protein more properties similar to that of glasses. While proteins function at ambient temperatures, cryogenic studies are necessary to understand the physics relevant for biology.

  17. Interfacial Protein-Protein Associations

    PubMed Central

    Langdon, Blake B.; Kastantin, Mark; Walder, Robert; Schwartz, Daniel K.

    2014-01-01

    While traditional models of protein adsorption focus primarily on direct protein-surface interactions, recent findings suggest that protein-protein interactions may play a central role. Using high-throughput intermolecular resonance energy transfer (RET) tracking, we directly observed dynamic, protein-protein associations of bovine serum albumin on poly(ethylene glycol) modified surfaces. The associations were heterogeneous and reversible, and associating molecules resided on the surface for longer times. The appearance of three distinct RET states suggested a spatially heterogeneous surface – with areas of high protein density (i.e. strongly-interacting clusters) coexisting with mobile monomers. Distinct association states exhibited characteristic behavior, i.e. partial-RET (monomer-monomer) associations were shorter-lived than complete-RET (protein-cluster) associations. While the fractional surface area covered by regions with high protein density (i.e. clusters) increased with increasing concentration, the distribution of contact times between monomers and clusters was independent of solution concentration, suggesting that associations were a local phenomenon, and independent of the global surface coverage. PMID:24274729

  18. Whey Protein

    MedlinePlus

    ... intolerance, for replacing or supplementing milk-based infant formulas, and for reversing weight loss and increasing glutathione ( ... allergic reactions compared to infants who receive standard formula. However, taking why protein might not be helpful ...

  19. Designed protein-protein association.

    PubMed

    Grueninger, Dirk; Treiber, Nora; Ziegler, Mathias O P; Koetter, Jochen W A; Schulze, Monika-Sarah; Schulz, Georg E

    2008-01-11

    The analysis of natural contact interfaces between protein subunits and between proteins has disclosed some general rules governing their association. We have applied these rules to produce a number of novel assemblies, demonstrating that a given protein can be engineered to form contacts at various points of its surface. Symmetry plays an important role because it defines the multiplicity of a designed contact and therefore the number of required mutations. Some of the proteins needed only a single side-chain alteration in order to associate to a higher-order complex. The mobility of the buried side chains has to be taken into account. Four assemblies have been structurally elucidated. Comparisons between the designed contacts and the results will provide useful guidelines for the development of future architectures. PMID:18187656

  20. 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.

  1. αvβ3- or α5β1-Integrin-Selective Peptidomimetics for Surface Coating.

    PubMed

    Mas-Moruno, Carlos; Fraioli, Roberta; Rechenmacher, Florian; Neubauer, Stefanie; Kapp, Tobias G; Kessler, Horst

    2016-06-13

    Engineering biomaterials with integrin-binding activity is a very powerful approach to promote cell adhesion, modulate cell behavior, and induce specific biological responses at the surface level. The aim of this Review is to illustrate the evolution of surface-coating molecules in this field: from peptides and proteins with relatively low integrin-binding activity and receptor selectivity to highly active and selective peptidomimetic ligands. In particular, we will bring into focus the difficult challenge of achieving selectivity between the two closely related integrin subtypes αvβ3 and α5β1. The functionalization of surfaces with such peptidomimetics opens the way for a new generation of highly specific cell-instructive surfaces to dissect the biological role of integrin subtypes and for application in tissue engineering and regenerative medicine. PMID:27258759

  2. 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

  3. 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...

  4. Integrin-binding elastin-like polypeptide as an in situ gelling delivery matrix enhances the therapeutic efficacy of adipose stem cells in healing full-thickness cutaneous wounds.

    PubMed

    Choi, Seong-Kyoon; Park, Jin-Kyu; Kim, Jung-Hee; Lee, Kyeong-Min; Kim, Enjoo; Jeong, Kyu-Shik; Jeon, Won Bae

    2016-09-10

    One crucial issue in stem cell therapy used for tissue repair is often the lack of selective carriers to deliver stem cells to the site of injury where the native extracellular matrix is pathologically damaged or lost. Therefore, it is necessary to develop a biomaterial that is permissive to stem cells and is suitable to replace injured or missing matrix. The major aim of this study is to investigate the potential of an RGD-containing elastin-like polypeptide (REP) with the structure TGPG[VGRGD(VGVPG)6]20WPC to engraft adipose stem cells (ASC) to full-thickness excisional wounds in mice. We implanted REP into the wound defects via body temperature-induced in situ aggregation. Engrafted REP exhibited a half-life of 2.6days in the wounds and did not elicit any pathological immune responses. REP itself significantly accelerated wound closure and reepithelialization and upregulated the expression of dermal tissue components. A combined administration of REP and ASC formed a hydrogel-like ASC/REP composite, which provided better neovascularization than the use of ASCs alone and increased the viability of transplanted ASC, improving overall wound healing. In vitro and in vivo mechanistic investigations suggested that REP enhances ASC survival at least in part via the Fak/Src adhesion-induced upregulation of Mek/Erk and PI3K/Akt survival pathways. We conclude that REP is a promising therapeutic agent for the improvement of stem cell-based therapy for enhanced tissue regeneration and repair. PMID:27393655

  5. Protein inference: A protein quantification perspective.

    PubMed

    He, Zengyou; Huang, Ting; Liu, Xiaoqing; Zhu, Peijun; Teng, Ben; Deng, Shengchun

    2016-08-01

    In mass spectrometry-based shotgun proteomics, protein quantification and protein identification are two major computational problems. To quantify the protein abundance, a list of proteins must be firstly inferred from the raw data. Then the relative or absolute protein abundance is estimated with quantification methods, such as spectral counting. Until now, most researchers have been dealing with these two processes separately. In fact, the protein inference problem can be regarded as a special protein quantification problem in the sense that truly present proteins are those proteins whose abundance values are not zero. Some recent published papers have conceptually discussed this possibility. However, there is still a lack of rigorous experimental studies to test this hypothesis. In this paper, we investigate the feasibility of using protein quantification methods to solve the protein inference problem. Protein inference methods aim to determine whether each candidate protein is present in the sample or not. Protein quantification methods estimate the abundance value of each inferred protein. Naturally, the abundance value of an absent protein should be zero. Thus, we argue that the protein inference problem can be viewed as a special protein quantification problem in which one protein is considered to be present if its abundance is not zero. Based on this idea, our paper tries to use three simple protein quantification methods to solve the protein inference problem effectively. The experimental results on six data sets show that these three methods are competitive with previous protein inference algorithms. This demonstrates that it is plausible to model the protein inference problem as a special protein quantification task, which opens the door of devising more effective protein inference algorithms from a quantification perspective. The source codes of our methods are available at: http://code.google.com/p/protein-inference/. PMID:26935399

  6. [The PAI-1 swing: microenvironment and cancer cell migration].

    PubMed

    Malo, Michel; Charrière-Bertrand, Cécile; Chettaoui, Chafika; Fabre-Guillevin, Elizabeth; Maquerlot, François; Lackmy, Alexandra; Vallée, Benoît; Delaplace, Franck; Barlovatz-Meimon, Georgia

    2006-12-01

    Cancer is a complex and dynamic process caused by a cellular dysfunction leading to a whole organ or even organism vital perturbation. To better understand this process, we need to study each one of the levels involved, which allows the scale change, and to integrate this knowledge. A matricellular protein, PAI-1, is able to induce in vitro cell behaviour modifications, morphological changes, and to promote cell migration. PAI-1 influences the mesenchymo-amaeboid transition. This matricellular protein should be considered as a potential 'launcher' of the metastatic process acting at the molecular, cellular, tissular levels and, as a consequence, at the organism's level. PMID:17126795

  7. 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

  8. Interfacing protein lysine acetylation and protein phosphorylation

    PubMed Central

    Tran, Hue T.; Uhrig, R. Glen; Nimick, Mhairi; Moorhead, Greg B.

    2012-01-01

    Recognition that different protein covalent modifications can operate in concert to regulate a single protein has forced us to re-think the relationship between amino acid side chain modifications and protein function. Results presented by Tran et al. 2012 demonstrate the association of a protein phosphatase (PP2A) with a histone/lysine deacetylase (HDA14) on plant microtubules along with a histone/lysine acetyltransferase (ELP3). This finding reveals a regulatory interface between two prevalent covalent protein modifications, protein phosphorylation and acetylation, emphasizing the integrated complexity of post-translational protein regulation found in nature. PMID:22827947

  9. Length, protein protein interactions, and complexity

    NASA Astrophysics Data System (ADS)

    Tan, Taison; Frenkel, Daan; Gupta, Vishal; Deem, Michael W.

    2005-05-01

    The evolutionary reason for the increase in gene length from archaea to prokaryotes to eukaryotes observed in large-scale genome sequencing efforts has been unclear. We propose here that the increasing complexity of protein-protein interactions has driven the selection of longer proteins, as they are more able to distinguish among a larger number of distinct interactions due to their greater average surface area. Annotated protein sequences available from the SWISS-PROT database were analyzed for 13 eukaryotes, eight bacteria, and two archaea species. The number of subcellular locations to which each protein is associated is used as a measure of the number of interactions to which a protein participates. Two databases of yeast protein-protein interactions were used as another measure of the number of interactions to which each S. cerevisiae protein participates. Protein length is shown to correlate with both number of subcellular locations to which a protein is associated and number of interactions as measured by yeast two-hybrid experiments. Protein length is also shown to correlate with the probability that the protein is encoded by an essential gene. Interestingly, average protein length and number of subcellular locations are not significantly different between all human proteins and protein targets of known, marketed drugs. Increased protein length appears to be a significant mechanism by which the increasing complexity of protein-protein interaction networks is accommodated within the natural evolution of species. Consideration of protein length may be a valuable tool in drug design, one that predicts different strategies for inhibiting interactions in aberrant and normal pathways.

  10. 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

  11. 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

  12. 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.

  13. 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.

  14. Protein folding, protein homeostasis, and cancer

    PubMed Central

    Van Drie, John H.

    2011-01-01

    Proteins fold into their functional 3-dimensional structures from a linear amino acid sequence. In vitro this process is spontaneous; while in vivo it is orchestrated by a specialized set of proteins, called chaperones. Protein folding is an ongoing cellular process, as cellular proteins constantly undergo synthesis and degradation. Here emerging links between this process and cancer are reviewed. This perspective both yields insights into the current struggle to develop novel cancer chemotherapeutics and has implications for future chemotherapy discovery. PMID:21272445

  15. Split-Protein Systems: Beyond Binary Protein-Protein Interactions

    PubMed Central

    Shekhawat, Sujan S.; Ghosh, Indraneel

    2011-01-01

    It has been estimated that 650,000 protein-protein interactions exist in the human interactome [1], a subset of all possible macromolecular partnerships that dictate life. Thus there is a continued need for the development of sensitive and user-friendly methods for cataloguing biomacromolecules in complex environments and for detecting their interactions, modifications, and cellular location. Such methods also allow for establishing differences in the interactome between a normal and diseased cellular state and for quantifying the outcome of therapeutic intervention. A promising approach for deconvoluting the role of macromolecular partnerships is split-protein reassembly, also called protein fragment complementation. This approach relies on the appropriate fragmentation of protein reporters, such as the green fluorescent protein or firefly luciferase, which when attached to possible interacting partners can reassemble and regain function, thereby confirming the partnership. Split-protein methods have been effectively utilized for detecting protein-protein interactions in cell-free systems, E. coli, yeast, mammalian cells, plants, and live animals. Herein, we present recent advances in engineering split-protein systems that allow for the rapid detection of ternary protein complexes, small molecule inhibitors, as well as a variety of macromolecules including nucleic acids, poly(ADP) ribose, and iron sulfur clusters. We also present advances that combine split-protein systems with chemical inducers of dimerization strategies that allow for regulating the activity of orthogonal split-proteases as well as aid in identifying enzyme inhibitors. Finally, we discuss autoinhibition strategies leading to turn-on sensors as well as future directions in split-protein methodology including possible therapeutic approaches. PMID:22070901

  16. Split-protein systems: beyond binary protein-protein interactions.

    PubMed

    Shekhawat, Sujan S; Ghosh, Indraneel

    2011-12-01

    It has been estimated that 650,000 protein-protein interactions exist in the human interactome (Stumpf et al., 2008), a subset of all possible macromolecular partnerships that dictate life. Thus there is a continued need for the development of sensitive and user-friendly methods for cataloguing biomacromolecules in complex environments and for detecting their interactions, modifications, and cellular location. Such methods also allow for establishing differences in the interactome between a normal and diseased cellular state and for quantifying the outcome of therapeutic intervention. A promising approach for deconvoluting the role of macromolecular partnerships is split-protein reassembly, also called protein fragment complementation. This approach relies on the appropriate fragmentation of protein reporters, such as the green fluorescent protein or firefly luciferase, which when attached to possible interacting partners can reassemble and regain function, thereby confirming the partnership. Split-protein methods have been effectively utilized for detecting protein-protein interactions in cell-free systems, Escherichia coli, yeast, mammalian cells, plants, and live animals. Herein, we present recent advances in engineering split-protein systems that allow for the rapid detection of ternary protein complexes, small molecule inhibitors, as well as a variety of macromolecules including nucleic acids, poly(ADP) ribose, and iron sulfur clusters. We also present advances that combine split-protein systems with chemical inducers of dimerization strategies that allow for regulating the activity of orthogonal split-proteases as well as aid in identifying enzyme inhibitors. Finally, we discuss autoinhibition strategies leading to turn-on sensors as well as future directions in split-protein methodology including possible therapeutic approaches. PMID:22070901

  17. 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 ...

  18. 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 ...

  19. Protein electrophoresis - serum

    MedlinePlus

    ... digestive tract to absorb proteins ( protein-losing enteropathy ) Malnutrition Kidney disorder called nephrotic syndrome Scarring of the ... may indicate: Abnormally low level of LDL cholesterol Malnutrition Increased gamma globulin proteins may indicate: Bone marrow ...

  20. Domains mediate protein-protein interactions and nucleate protein assemblies.

    PubMed

    Costa, S; Cesareni, G

    2008-01-01

    Cell physiology is governed by an intricate mesh of physical and functional links among proteins, nucleic acids and other metabolites. The recent information flood coming from large-scale genomic and proteomic approaches allows us to foresee the possibility of compiling an exhaustive list of the molecules present within a cell, enriched with quantitative information on concentration and cellular localization. Moreover, several high-throughput experimental and computational techniques have been devised to map all the protein interactions occurring in a living cell. So far, such maps have been drawn as graphs where nodes represent proteins and edges represent interactions. However, this representation does not take into account the intrinsically modular nature of proteins and thus fails in providing an effective description of the determinants of binding. Since proteins are composed of domains that often confer on proteins their binding capabilities, a more informative description of the interaction network would detail, for each pair of interacting proteins in the network, which domains mediate the binding. Understanding how protein domains combine to mediate protein interactions would allow one to add important features to the protein interaction network, making it possible to discriminate between simultaneously occurring and mutually exclusive interactions. This objective can be achieved by experimentally characterizing domain recognition specificity or by analyzing the frequency of co-occurring domains in proteins that do interact. Such approaches allow gaining insights on the topology of complexes with unknown three-dimensional structure, thus opening the prospect of adopting a more rational strategy in developing drugs designed to selectively target specific protein interactions. PMID:18491061

  1. Drugging Membrane Protein Interactions.

    PubMed

    Yin, Hang; Flynn, Aaron D

    2016-07-11

    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 cells 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 of 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

  2. Protein sensing with engineered protein nanopores*

    PubMed Central

    Mohammad, Mohammad M.; Movileanu, Liviu

    2013-01-01

    The use of nanopores is a powerful new frontier in single-molecule sciences. Nanopores have been used effectively in exploring various biophysical features of small polypeptides and proteins, such as their folding state and structure, ligand interactions, and enzymatic activity. In particular, the α-hemolysin protein pore (αHL) has been used extensively for the detection, characterization and analysis of polypeptides, because this protein nanopore is highly robust, versatile and tractable under various experimental conditions. Inspired by the mechanisms of protein translocation across the outer membrane translocases of mitochondria, we have shown the ability to use nanopore-probe techniques in controlling a single protein using engineered αHL pores. Here, we provide a detailed protocol for the preparation of αHL protein nanopores. Moreover, we demonstrate that placing attractive electrostatic traps is instrumental in tackling single-molecule stochastic sensing of folded proteins. PMID:22528256

  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. Functional role of periostin in development and wound repair: implications for connective tissue disease

    PubMed Central

    2008-01-01

    Integrity of the extracellular matrix (ECM) is essential for maintaining the normal structure and function of connective tissues. ECM is secreted locally by cells and organized into a complex meshwork providing physical support to cells, tissues, and organs. Initially thought to act only as a scaffold, the ECM is now known to provide a myriad of signals to cells regulating all aspects of their phenotype from morphology to differentiation. Matricellular proteins are a class of ECM related molecules defined through their ability to modulate cell–matrix interactions. Matricellular proteins are expressed at high levels during development, but typically only appear in postnatal tissue in wound repair or disease, where their levels increase substantially. Members of the CCN family, tenascin-C, osteopontin, secreted protein acidic rich in cysteine (SPARC), bone sialoprotein, thrombospondins, and galectins have all been classed as matricellular proteins. Periostin, a 90 kDa secreted homophilic cell adhesion protein, was recently added to matricellular class of proteins based on its expression pattern and function during development as well as in wound repair. Periostin is expressed in connective tissues including the periodontal ligament, tendons, skin and bone, and is also prominent in neoplastic tissues, cardiovascular disease, as well as in connective tissue wound repair. This review will focus on the functional role of periostin in tissue physiology. Fundamentally, it appears that periostin influences cell behaviour as well as collagen fibrillogenesis, and therefore exerts control over the structural and functional properties of connective tissues in both health and disease. Periostin is a novel matricellular protein with close homology to Drosophila fasciclin 1. In this review, the functional role of periostin is discussed in the context of connective tissue physiology, in development, disease, and wound repair. PMID:18642132

  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. Sorghum and millet proteins

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Sorghum and millet proteins are an important source of dietary protein for significant numbers of people living throughout Africa and parts of Asia. Compared to other food proteins, such as those found in milk, eggs and wheat, little is known about the functionality of sorghum and millet proteins. ...

  8. Whey protein fractionation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Concentrated whey protein products from cheese whey, such as whey protein concentrate (WPC) and whey protein isolate (WPI), contain more than seven different types of proteins: alpha-lactalbumin (alpha-LA), beta-lactoglobulin (beta-LG), bovine serum albumin (BSA), immunoglobulins (Igs), lactoferrin ...

  9. Protein in diet

    MedlinePlus

    ... protein. The basic structure of protein is a chain of amino acids. You need protein in your diet to help your body repair cells and make new ones. Protein is also important for growth and development in children, teens, and pregnant women.

  10. Techniques in protein methylation.

    PubMed

    Lee, Jaeho; Cheng, Donghang; Bedford, Mark T

    2004-01-01

    Proteins can be methylated on the side-chain nitrogens of arginine and lysine residues or on carboxy-termini. Protein methylation is a way of subtly changing the primary sequence of a peptide so that it can encode more information. This common posttranslational modification is implicated in the regulation of a variety of processes including protein trafficking, transcription and protein-protein interactions. In this chapter, we will use the arginine methyltransferases to illustrate different approaches that have been developed to assess protein methylation. Both in vivo and in vitro methylation techniques are described, and the use of small molecule inhibitors of protein methylation will be demonstrated. PMID:15173617

  11. Biochemical Approaches for Discovering Protein-Protein Interactions

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Protein-protein interactions or protein complexes are indigenous to nearly all cellular processes, ranging from metabolism to structure. Elucidating both individual protein associations and complex protein interaction networks, while challenging, is an essential goal of functional genomics. For ex...

  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. [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

  14. 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.

  15. 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

  16. Surface Mediated Protein Disaggregation

    NASA Astrophysics Data System (ADS)

    Radhakrishna, Mithun; Kumar, Sanat K.

    2014-03-01

    Preventing protein aggregation is of both biological and industrial importance. Biologically these aggregates are known to cause amyloid type diseases like Alzheimer's and Parkinson's disease. Protein aggregation leads to reduced activity of the enzymes in industrial applications. Inter-protein interactions between the hydrophobic residues of the protein are known to be the major driving force for protein aggregation. In the current paper we show how surface chemistry and curvature can be tuned to mitigate these inter-protein interactions. Our results calculated in the framework of the Hydrophobic-Polar (HP) lattice model show that, inter-protein interactions can be drastically reduced by increasing the surface hydrophobicity to a critical value corresponding to the adsorption transition of the protein. At this value of surface hydrophobicity, proteins lose inter-protein contacts to gain surface contacts and thus the surface helps in reducing the inter-protein interactions. Further, we show that the adsorption of the proteins inside hydrophobic pores of optimal sizes are most efficient both in reducing inter-protein contacts and simultaneously retaining most of the native-contacts due to strong protein-surface interactions coupled with stabilization due to the confinement. Department of Energy (Grant No DE-FG02-11ER46811).

  17. PINT: Protein-protein Interactions Thermodynamic Database.

    PubMed

    Kumar, M D Shaji; Gromiha, M Michael

    2006-01-01

    The first release of Protein-protein Interactions Thermodynamic Database (PINT) contains >1500 data of several thermodynamic parameters along with sequence and structural information, experimental conditions and literature information. Each entry contains numerical data for the free energy change, dissociation constant, association constant, enthalpy change, heat capacity change and so on of the interacting proteins upon binding, which are important for understanding the mechanism of protein-protein interactions. PINT also includes the name and source of the proteins involved in binding, their Protein Information Resource, SWISS-PROT and Protein Data Bank (PDB) codes, secondary structure and solvent accessibility of residues at mutant positions, measuring methods, experimental conditions, such as buffers, ions and additives, and literature information. A WWW interface facilitates users to search data based on various conditions, feasibility to select the terms for output and different sorting options. Further, PINT is cross-linked with other related databases, PIR, SWISS-PROT, PDB and NCBI PUBMED literature database. The database is freely available at http://www.bioinfodatabase.com/pint/index.html. PMID:16381844

  18. 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

  19. 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

  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 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 ...

  2. Protein electrophoresis - urine

    MedlinePlus

    ... nephropathy Kidney failure Multiple myeloma Nephrotic syndrome Acute urinary tract infection Risks There are no risks associated with this ... Primary amyloidosis Protein in diet Protein urine test Urinary tract infection - adults Update Date 5/29/2014 Updated by: ...

  3. [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

  4. 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 ...

  5. Hydrodynamic effects in proteins

    NASA Astrophysics Data System (ADS)

    Szymczak, Piotr; Cieplak, Marek

    2011-01-01

    Experimental and numerical results pertaining to flow-induced effects in proteins are reviewed. Special emphasis is placed on shear-induced unfolding and on the role of solvent mediated hydrodynamic interactions in the conformational transitions in proteins.

  6. Hydrodynamic effects in proteins.

    PubMed

    Szymczak, Piotr; Cieplak, Marek

    2011-01-26

    Experimental and numerical results pertaining to flow-induced effects in proteins are reviewed. Special emphasis is placed on shear-induced unfolding and on the role of solvent mediated hydrodynamic interactions in the conformational transitions in proteins. PMID:21406855

  7. Understanding protein folding: small proteins in silico.

    PubMed

    Zimmermann, Olav; Hansmann, Ulrich H E

    2008-01-01

    Recent improvements in methodology and increased computer power now allow atomistic computer simulations of protein folding. We briefly review several advanced Monte Carlo algorithms that have contributed to this development. Details of folding simulations of three designed mini proteins are shown. Adding global translations and rotations has allowed us to handle multiple chains and to simulate the aggregation of six beta-amyloid fragments. In a different line of research we have developed several algorithms to predict local features from sequence. In an outlook we sketch how such biasing could extend the application spectrum of Monte Carlo simulations to structure prediction of larger proteins. PMID:18036571

  8. Imaging Protein-protein Interactions in vivo

    PubMed Central

    Seegar, Tom; Barton, William

    2010-01-01

    Protein-protein interactions are a hallmark of all essential cellular processes. However, many of these interactions are transient, or energetically weak, preventing their identification and analysis through traditional biochemical methods such as co-immunoprecipitation. In this regard, the genetically encodable fluorescent proteins (GFP, RFP, etc.) and their associated overlapping fluorescence spectrum have revolutionized our ability to monitor weak interactions in vivo using Förster resonance energy transfer (FRET)1-3. Here, we detail our use of a FRET-based proximity assay for monitoring receptor-receptor interactions on the endothelial cell surface. PMID:20972411

  9. CSF myelin basic protein

    MedlinePlus

    CSF myelin basic protein is a test to measure the level of myelin basic protein (MBP) in the cerebrospinal fluid (CSF). The CSF ... less than 4 ng/mL of myelin basic protein in the CSF. Normal value ranges may vary ...

  10. 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…

  11. Palmitoylation of Hedgehog proteins.

    PubMed

    Buglino, John A; Resh, Marilyn D

    2012-01-01

    Hedgehog (Hh) proteins are secreted signaling proteins that contain amide-linked palmitate at the N-terminus and cholesterol at the C-terminus. Palmitoylation of Hh proteins is critical for effective long- and short-range signaling. The palmitoylation reaction occurs during transit of Hh through the secretory pathway, most likely in the lumen of the ER. Attachment of palmitate to Hh proteins is independent of cholesterol modification and autoprocessing and is catalyzed by Hhat (Hedgehog acyltransferase). Hhat is a member of the membrane bound O-acyltransferase (MBOAT) family, a subgroup of multipass membrane proteins that catalyze transfer of fatty acyl groups to lipids and proteins. Several classes of secreted proteins have recently been shown to be substrates for MBOAT acyltransferases, including Hh proteins and Spitz (palmitoylated by Hhat), Wg/Wnt proteins (modified with palmitate and/or palmitoleate by Porcupine) and ghrelin (octanoylated by ghrelin O-acyltransferase). These findings highlight protein fatty acylation as a mechanism that not only influences membrane binding of intracellular proteins but also regulates the signaling range and efficacy of secreted proteins. PMID:22391306

  12. Protein electrophoresis - serum

    MedlinePlus

    Normal value ranges are: Total protein: 6.4 to 8.3 g/dL (grams per deciliter) Albumin: 3.5 to 5.0 g/dL Alpha-1 ... Decreased total protein may indicate: Abnormal loss of protein from the digestive tract or the inability of the digestive tract ...

  13. 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 = ...

  14. 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…

  15. 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...

  16. 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.

  17. Endothelial Cell Response to Chemical, Biological, and Physical Cues in Bioactive Hydrogels

    PubMed Central

    Browning, Mary Beth; Guiza, Viviana; Russell, Brooke; Rivera, Jose; Cereceres, Stacy; Höök, Magnus; Hahn, Mariah S.

    2014-01-01

    The highly tunable biological, chemical, and physical properties of bioactive hydrogels enable their use in an array of tissue engineering and drug delivery applications. Systematic modulation of these properties can be used to elucidate key cell–material interactions to improve therapeutic effects. For example, the rate and extent of endothelialization are critical to the long-term success of many blood-contacting devices. To this end, we have developed a bioactive hydrogel that could be used as coating on cardiovascular devices to enhance endothelial cell (EC) adhesion and migration. The current work investigates the relative impact of hydrogel variables on key endothelialization processes. The bioactive hydrogel is based on poly(ethylene glycol) (PEG) and a streptococcal collagen-like (Scl2-2) protein that has been modified with integrin α1β1 and α2β1 binding sites. The use of PEG hydrogels allows for incorporation of specific bioactive cues and independent manipulation of scaffold properties. The selective integrin binding of Scl2-2 was compared to more traditional collagen-modified PEG hydrogels to determine the effect of integrin binding on cell behavior. Protein functionalization density, protein concentration, and substrate modulus were independently tuned with both Scl2-2 and collagen to determine the effect of each variable on EC adhesion, spreading, and migration. The findings here demonstrate that increasing substrate modulus, decreasing functionalization density, and increasing protein concentration can be utilized to increase EC adhesion and migration. Additionally, PEG-Scl2-2 hydrogels had higher migration speeds and proliferation over 1 week compared with PEG-collagen gels, demonstrating that selective integrin binding can be used to enhance cell–material interactions. Overall, these studies contribute to the understanding of the effects of matrix cues on EC interactions and demonstrate the strong potential of PEG-Scl2-2 hydrogels to promote

  18. Protein - Which is Best?

    PubMed

    Hoffman, Jay R; Falvo, Michael J

    2004-09-01

    Protein intake that exceeds the recommended daily allowance is widely accepted for both endurance and power athletes. However, considering the variety of proteins that are available much less is known concerning the benefits of consuming one protein versus another. The purpose of this paper is to identify and analyze key factors in order to make responsible recommendations to both the general and athletic populations. Evaluation of a protein is fundamental in determining its appropriateness in the human diet. Proteins that are of inferior content and digestibility are important to recognize and restrict or limit in the diet. Similarly, such knowledge will provide an ability to identify proteins that provide the greatest benefit and should be consumed. The various techniques utilized to rate protein will be discussed. Traditionally, sources of dietary protein are seen as either being of animal or vegetable origin. Animal sources provide a complete source of protein (i.e. containing all essential amino acids), whereas vegetable sources generally lack one or more of the essential amino acids. Animal sources of dietary protein, despite providing a complete protein and numerous vitamins and minerals, have some health professionals concerned about the amount of saturated fat common in these foods compared to vegetable sources. The advent of processing techniques has shifted some of this attention and ignited the sports supplement marketplace with derivative products such as whey, casein and soy. Individually, these products vary in quality and applicability to certain populations. The benefits that these particular proteins possess are discussed. In addition, the impact that elevated protein consumption has on health and safety issues (i.e. bone health, renal function) are also reviewed. Key PointsHigher protein needs are seen in athletic populations.Animal proteins is an important source of protein, however potential health concerns do exist from a diet of protein

  19. 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.

  20. 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.

  1. 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.

  2. 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.

  3. 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. PMID:26836410

  4. 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

  5. Mechanism of protein decarbonylation.

    PubMed

    Wong, Chi-Ming; Marcocci, Lucia; Das, Dividutta; Wang, Xinhong; Luo, Haibei; Zungu-Edmondson, Makhosazane; Suzuki, Yuichiro J

    2013-12-01

    Ligand/receptor stimulation of cells promotes protein carbonylation that is followed by the decarbonylation process, which might involve thiol-dependent reduction (C.M. Wong et al., Circ. Res. 102:301-318; 2008). This study further investigated the properties of this protein decarbonylation mechanism. We found that the thiol-mediated reduction of protein carbonyls is dependent on heat-labile biologic components. Cysteine and glutathione were efficient substrates for decarbonylation. Thiols decreased the protein carbonyl content, as detected by 2,4-dinitrophenylhydrazine, but not the levels of malondialdehyde or 4-hydroxynonenal protein adducts. Mass spectrometry identified proteins that undergo thiol-dependent decarbonylation, which include peroxiredoxins. Peroxiredoxin-2 and -6 were carbonylated and subsequently decarbonylated in response to the ligand/receptor stimulation of cells. siRNA knockdown of glutaredoxin inhibited the decarbonylation of peroxiredoxin. These results strengthen the concept that thiol-dependent decarbonylation defines the kinetics of protein carbonylation signaling. PMID:24044890

  6. 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.

  7. 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.

  8. 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.

  9. Phage display of proteins.

    PubMed

    Kościelska, K; Kiczak, L; Kasztura, M; Wesołowska, O; Otlewski, J

    1998-01-01

    In recent years the phage display approach has become an increasingly popular method in protein research. This method enables the presentation of large peptide and protein libraries on the surface of phage particles from which molecules of desired functional property(ies) can be rapidly selected. The great advantage of this method is a direct linkage between an observed phenotype and encapsulated genotype, which allows fast determination of selected sequences. The phage display approach is a powerful tool in generating highly potent biomolecules, including: search for specific antibodies, determining enzyme specificity, exploring protein-protein and protein-DNA interactions, minimizing proteins, introducing new functions into different protein scaffolds, and searching sequence space of protein folding. In this article many examples are given to illustrate that this technique can be used in different fields of protein science. The phage display has a potential of the natural evolution and its possibilities are far beyond rational prediction. Assuming that we can design the selection agents and conditions we should be able to engineer any desired protein function or feature. PMID:9918498

  10. 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

  11. 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

  12. Protein-protein docking with backbone flexibility.

    PubMed

    Wang, Chu; Bradley, Philip; Baker, David

    2007-10-19

    Computational protein-protein docking methods currently can create models with atomic accuracy for protein complexes provided that the conformational changes upon association are restricted to the side chains. However, it remains very challenging to account for backbone conformational changes during docking, and most current methods inherently keep monomer backbones rigid for algorithmic simplicity and computational efficiency. Here we present a reformulation of the Rosetta docking method that incorporates explicit backbone flexibility in protein-protein docking. The new method is based on a "fold-tree" representation of the molecular system, which seamlessly integrates internal torsional degrees of freedom and rigid-body degrees of freedom. Problems with internal flexible regions ranging from one or more loops or hinge regions to all of one or both partners can be readily treated using appropriately constructed fold trees. The explicit treatment of backbone flexibility improves both sampling in the vicinity of the native docked conformation and the energetic discrimination between near-native and incorrect models. PMID:17825317

  13. Energy design for protein-protein interactions

    NASA Astrophysics Data System (ADS)

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

    2011-08-01

    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.

  14. Mechanisms Regulating Protein Localization.

    PubMed

    Bauer, Nicholas C; Doetsch, Paul W; Corbett, Anita H

    2015-10-01

    Cellular functions are dictated by protein content and activity. There are numerous strategies to regulate proteins varying from modulating gene expression to post-translational modifications. One commonly used mode of regulation in eukaryotes is targeted localization. By specifically redirecting the localization of a pool of existing protein, cells can achieve rapid changes in local protein function. Eukaryotic cells have evolved elegant targeting pathways to direct proteins to the appropriate cellular location or locations. Here, we provide a general overview of these localization pathways, with a focus on nuclear and mitochondrial transport, and present a survey of the evolutionarily conserved regulatory strategies identified thus far. We end with a description of several specific examples of proteins that exploit localization as an important mode of regulation. PMID:26172624

  15. 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

  16. Outer membrane protein purification.

    PubMed

    Arigita, C; Jiskoot, W; Graaf, M R; Kersten, G F

    2001-01-01

    The major outer membrane proteins (OMPs) from Neisseria meningitidis, which are expressed at high levels, are subdivided in five classes based on molecular weight (1,2) (see Table 1). Table 1 Major Meningococcal Outer-Membrane Proteins Outer-membrane proteins Name Molecular maass Function/characteristics Class 1 PorA 44-47 kDa Porin Class 2/3 PorB 37-42 kDa Porin Class 4 Rmp Reductionmodifiableprotein, unknown Class 5 Opa 26-30 kDa Adhesion,opacity protein Opc 25 kDa Invasion, opacity protein Iron-regulated proteins Mirp 37 kDa Iron acquisition (?);majoriron-regulatedprotein FrpB 70 kDa Ferric enterobactin receptor (also FetA) Adapted from ref. (1). PMID:21336748

  17. 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

  18. 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

  19. 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

  20. Elastic proteins and elastomeric protein alloys.

    PubMed

    Aghaei-Ghareh-Bolagh, Behnaz; Mithieux, Suzanne M; Weiss, Anthony S

    2016-06-01

    The elastomeric proteins elastin and resilin have been used extensively in the fabrication of biomaterials for tissue engineering applications due to their unique mechanical and biological properties. Tropoelastin is the soluble monomer component of elastin. Tropoelastin and resilin are both highly elastic with high resilience, substantial extensibility, high durability and low energy loss, which makes them excellent candidates for the fabrication of elastic tissues that demand regular and repetitive movement like the skin, lung, blood vessels, muscles and vocal folds. Combinations of these proteins with silk fibroin further enhance their biomechanical and biological properties leading to a new class of protein alloy materials with versatile properties. In this review, the properties of tropoelastin-based and resilin-based biomaterials with and without silk are described in concert with examples of their applications in tissue engineering. PMID:26780495

  1. Nanotopography Facilitates in Vivo Transdermal Delivery of High Molecular Weight Therapeutics through an Integrin-Dependent Mechanism

    PubMed Central

    Walsh, Laura; Ryu, Jubin; Bock, Suzanne; Koval, Michael; Mauro, Theodora; Ross, Russell; Desai, Tejal

    2015-01-01

    Transdermal delivery of therapeutics is restricted by narrow limitations on size and hydrophobicity. Nanotopography has been shown to significantly enhance high molecular weight paracellular transport in vitro. Herein, we demonstrate for the first time that nanotopography applied to microneedles significantly enhances transdermal delivery of etanercept, a 150 kD therapeutic, in both rats and rabbits. We further show that this effect is mediated by remodeling of the tight junction proteins initiated via integrin binding to the nanotopography, followed by phosphorylation of myosin light chain (MLC) and activation of the actomyosin complex, which in turn increase paracellular permeability. PMID:25790174

  2. 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)

  3. 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. PMID:27026395

  4. 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

  5. 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.

  6. 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.

  7. Protein-protein interactions as drug targets.

    PubMed

    Skwarczynska, Malgorzata; Ottmann, Christian

    2015-10-01

    Modulation of protein-protein interactions (PPIs) is becoming increasingly important in drug discovery and chemical biology. While a few years ago this 'target class' was deemed to be largely undruggable an impressing number of publications and success stories now show that targeting PPIs with small, drug-like molecules indeed is a feasible approach. Here, we summarize the current state of small-molecule inhibition and stabilization of PPIs and review the active molecules from a structural and medicinal chemistry angle, especially focusing on the key examples of iNOS, LFA-1 and 14-3-3. PMID:26510391

  8. 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.

  9. 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.

  10. Consensus protein design.

    PubMed

    Porebski, Benjamin T; Buckle, Ashley M

    2016-07-01

    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

  11. Prediction of protein-protein interactions based on protein-protein correlation using least squares regression.

    PubMed

    Huang, De-Shuang; Zhang, Lei; Han, Kyungsook; Deng, Suping; Yang, Kai; Zhang, Hongbo

    2014-01-01

    In order to transform protein sequences into the feature vectors, several works have been done, such as computing auto covariance (AC), conjoint triad (CT), local descriptor (LD), moran autocorrelation (MA), normalized moreaubroto autocorrelation (NMB) and so on. In this paper, we shall adopt these transformation methods to encode the proteins, respectively, where AC, CT, LD, MA and NMB are all represented by '+' in a unified manner. A new method, i.e. the combination of least squares regression with '+' (abbreviated as LSR(+)), will be introduced for encoding a protein-protein correlation-based feature representation and an interacting protein pair. Thus there are totally five different combinations for LSR(+), i.e. LSRAC, LSRCT, LSRLD, LSRMA and LSRNMB. As a result, we combined a support vector machine (SVM) approach with LSR(+) to predict protein-protein interactions (PPI) and PPI networks. The proposed method has been applied on four datasets, i.e. Saaccharomyces cerevisiae, Escherichia coli, Homo sapiens and Caenorhabditis elegans. The experimental results demonstrate that all LSR(+) methods outperform many existing representative algorithms. Therefore, LSR(+) is a powerful tool to characterize the protein-protein correlations and to infer PPI, whilst keeping high performance on prediction of PPI networks. PMID:25059329

  12. 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.

  13. PIC: Protein Interactions Calculator.

    PubMed

    Tina, K G; Bhadra, R; Srinivasan, N

    2007-07-01

    Interactions within a protein structure and interactions between proteins in an assembly are essential considerations in understanding molecular basis of stability and functions of proteins and their complexes. There are several weak and strong interactions that render stability to a protein structure or an assembly. Protein Interactions Calculator (PIC) is a server which, given the coordinate set of 3D structure of a protein or an assembly, computes various interactions such as disulphide bonds, interactions between hydrophobic residues, ionic interactions, hydrogen bonds, aromatic-aromatic interactions, aromatic-sulphur interactions and cation-pi interactions within a protein or between proteins in a complex. Interactions are calculated on the basis of standard, published criteria. The identified interactions between residues can be visualized using a RasMol and Jmol interface. The advantage with PIC server is the easy availability of inter-residue interaction calculations in a single site. It also determines the accessible surface area and residue-depth, which is the distance of a residue from the surface of the protein. User can also recognize specific kind of interactions, such as apolar-apolar residue interactions or ionic interactions, that are formed between buried or exposed residues or near the surface or deep inside. PMID:17584791

  14. 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

  15. Engineering therapeutic protein disaggregases.

    PubMed

    Shorter, James

    2016-05-15

    Therapeutic agents are urgently required to cure several common and fatal neurodegenerative disorders caused by protein misfolding and aggregation, including amyotrophic lateral sclerosis (ALS), Parkinson's disease (PD), and Alzheimer's disease (AD). Protein disaggregases that reverse protein misfolding and restore proteins to native structure, function, and localization could mitigate neurodegeneration by simultaneously reversing 1) any toxic gain of function of the misfolded form and 2) any loss of function due to misfolding. Potentiated variants of Hsp104, a hexameric AAA+ ATPase and protein disaggregase from yeast, have been engineered to robustly disaggregate misfolded proteins connected with ALS (e.g., TDP-43 and FUS) and PD (e.g., α-synuclein). However, Hsp104 has no metazoan homologue. Metazoa possess protein disaggregase systems distinct from Hsp104, including Hsp110, Hsp70, and Hsp40, as well as HtrA1, which might be harnessed to reverse deleterious protein misfolding. Nevertheless, vicissitudes of aging, environment, or genetics conspire to negate these disaggregase systems in neurodegenerative disease. Thus, engineering potentiated human protein disaggregases or isolating small-molecule enhancers of their activity could yield transformative therapeutics for ALS, PD, and AD. PMID:27255695

  16. Cellulose synthase interacting protein

    PubMed Central

    Somerville, Chris

    2010-01-01

    Cellulose is the most abundant biopolymer on earth. The great abundance of cellulose places it at the forefront as a primary source of biomass for renewable biofuels. However, the knowledge of how plant cells make cellulose remains very rudimentary. Cellulose microfibrils are synthesized at the plasma membrane by hexameric protein complexes, also known as cellulose synthase complexes. The only known components of cellulose synthase complexes are cellulose synthase (CESA) proteins until the recent identification of a novel component. CSI1, which encodes CESA interacting protein 1 (CSI1) in Arabidopsis. CSI1, as the first non-CESA proteins associated with cellulose synthase complexes, opens up many opportunities. PMID:21150290

  17. 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

  18. 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

  19. Engineering therapeutic protein disaggregases

    PubMed Central

    Shorter, James

    2016-01-01

    Therapeutic agents are urgently required to cure several common and fatal neurodegenerative disorders caused by protein misfolding and aggregation, including amyotrophic lateral sclerosis (ALS), Parkinson’s disease (PD), and Alzheimer’s disease (AD). Protein disaggregases that reverse protein misfolding and restore proteins to native structure, function, and localization could mitigate neurodegeneration by simultaneously reversing 1) any toxic gain of function of the misfolded form and 2) any loss of function due to misfolding. Potentiated variants of Hsp104, a hexameric AAA+ ATPase and protein disaggregase from yeast, have been engineered to robustly disaggregate misfolded proteins connected with ALS (e.g., TDP-43 and FUS) and PD (e.g., α-synuclein). However, Hsp104 has no metazoan homologue. Metazoa possess protein disaggregase systems distinct from Hsp104, including Hsp110, Hsp70, and Hsp40, as well as HtrA1, which might be harnessed to reverse deleterious protein misfolding. Nevertheless, vicissitudes of aging, environment, or genetics conspire to negate these disaggregase systems in neurodegenerative disease. Thus, engineering potentiated human protein disaggregases or isolating small-molecule enhancers of their activity could yield transformative therapeutics for ALS, PD, and AD. PMID:27255695

  20. Ultrafiltration of pegylated proteins

    NASA Astrophysics Data System (ADS)

    Molek, Jessica R.

    There is considerable clinical interest in the use of "second-generation" therapeutics produced by conjugation of a native protein with various polymers including polyethylene glycol (PEG). PEG--protein conjugates, so-called PEGylated proteins, can exhibit enhanced stability, half-life, and bioavailability. One of the challenges in the commercial production of PEGylated proteins is the purification required to remove unreacted polymer, native protein, and in many cases PEGylated proteins with nonoptimal degrees of conjugation. The overall objective of this thesis was to examine the use of ultrafiltration for the purification of PEGylated proteins. This included: (1) analysis of size-based separation of PEGylated proteins using conventional ultrafiltration membranes, (2) use of electrically-charged membranes to exploit differences in electrostatic interactions, and (3) examination of the effects of PEGylation on protein fouling. The experimental results were analyzed using appropriate theoretical models, with the underlying physical properties of the PEGylated proteins evaluated using size exclusion chromatography, capillary electrophoresis, dynamic light scattering, and reverse phase chromatography. PEGylated proteins were produced by covalent attachment of activated PEG to a protein via primary amines on the lysine residues. A simple model was developed for the reaction kinetics, which was used to explore the effect of reaction conditions and mode of operation on the distribution of PEGylated products. The effective size of the PEGylated proteins was evaluated using size exclusion chromatography, with appropriate correlations developed for the size in terms of the molecular weight of the native protein and attached PEG. The electrophoretic mobility of the PEGylated proteins were evaluated by capillary electrophoresis with the data in good agreement with a simple model accounting for the increase in protein size and the reduction in the number of protonated amine

  1. 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

  2. Binding Efficiency of Protein-Protein Complexes

    PubMed Central

    Day, Eric S.; Cote, Shaun M.; Whitty, Adrian

    2012-01-01

    We examine the relationship between binding affinity and interface size for reversible protein-protein interactions (PPI), using cytokines from the tumor necrosis factor (TNF) superfamily and their receptors as a test case. Using surface plasmon resonance, we measured single-site binding affinities for the large receptor TNFR1 binding to its ligands TNFα (KD = 1.4 ± 0.4 nM) and lymphotoxin-α (KD = 50 ± 10 nM), and also for the small receptor Fn14 binding to TWEAK (KD = 70 ± 10 nM). We additionally assembled data for all other TNF/TNFR family complexes for which reliable single site binding affinities have been reported. We used these values to calculate the binding efficiency – defined as binding energy per Å2 of surface area buried at the contact interface – for the nine of these complexes for which co-crystal structures are available, and compared the results to those for a set of 144 protein-protein complexes with published affinity values. The results show that the most efficient PPI complexes generate ~20 cal.mol−1/Å2 of binding energy. A minimum contact area of ~500 Å2 is required for a stable complex, required to generate sufficient interaction energy to pay the entropic cost of co-localizing two proteins from 1 M solution. The most compact and efficient TNF/TNFR complex was BAFF/BR3, which achieved ~80% of the maximum achievable binding efficiency. Other small receptors also gave high binding efficiencies, while the larger receptors generated only 44-49% of this limit despite interacting primarily through just a single small domain. The results provide new insight into how much binding energy can be generated by a PPI interface of a given size, and establish a quantitative method to predict how large a natural or engineered contact interface must be to achieve a given level of binding affinity. PMID:23088250

  3. Protein Attachment on Nanodiamonds.

    PubMed

    Lin, Chung-Lun; Lin, Cheng-Huang; Chang, Huan-Cheng; Su, Meng-Chih

    2015-07-16

    A recent advance in nanotechnology is the scale-up production of small and nonaggregated diamond nanoparticles suitable for biological applications. Using detonation nanodiamonds (NDs) with an average diameter of ∼4 nm as the adsorbents, we have studied the static attachment of three proteins (myoglobin, bovine serum albumin, and insulin) onto the nanoparticles by optical spectroscopy, mass spectrometry, and dynamic light scattering, and electrophoretic zeta potential measurements. Results show that the protein surface coverage is predominantly determined by the competition between protein-protein and protein-ND interactions, giving each protein a unique and characteristic structural configuration in its own complex. Specifically, both myoglobin and bovine serum albumin show a Langmuir-type adsorption behavior, forming 1:1 complexes at saturation, whereas insulin folds into a tightly bound multimer before adsorption. The markedly different adsorption patterns appear to be independent of the protein concentration and are closely related to the affinity of the individual proteins for the NDs. The present study provides a fundamental understanding for the use of NDs as a platform for nanomedical drug delivery. PMID:25815400

  4. 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

  5. Proteins and Amino Acids

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Proteins are the most abundant substances in living organisms and cells. All proteins are constructed from the same twenty amino acids that are linked together by covalent bonds. Shorter chains of two or more amino acids can be linked by covalent bonds to form polypeptides. There are twenty amino...

  6. Proteins and glasses

    SciTech Connect

    Frauenfelder, H.

    1997-12-31

    The structure, the energy landscape, and the dynamics of proteins and glasses are similar. Both types of systems display characteristic nonexponential time dependencies of relaxation phenomena. Experiments suggest that both, proteins and glasses, are heterogeneous and that this fact causes the observed time dependence. This result is discussed in terms of the rough energy landscape characteristic of complex systems.

  7. Synthesis of Lipidated Proteins.

    PubMed

    Mejuch, Tom; Waldmann, Herbert

    2016-08-17

    Protein lipidation is one of the major post-translational modifications (PTM) of proteins. The attachment of the lipid moiety frequently determines the localization and the function of the lipoproteins. Lipidated proteins participate in many essential biological processes in eukaryotic cells, including vesicular trafficking, signal transduction, and regulation of the immune response. Malfunction of these cellular processes usually leads to various diseases such as cancer. Understanding the mechanism of cellular signaling and identifying the protein-protein and protein-lipid interactions in which the lipoproteins are involved is a crucial task. To achieve these goals, fully functional lipidated proteins are required. However, access to lipoproteins by means of standard expression is often rather limited. Therefore, semisynthetic methods, involving the synthesis of lipidated peptides and their subsequent chemoselective ligation to yield full-length lipoproteins, were developed. In this Review we summarize the commonly used methods for lipoprotein synthesis and the development of the corresponding chemoselective ligation techniques. Several key studies involving full-length semisynthetic lipidated Ras, Rheb, and LC3 proteins are presented. PMID:27444727

  8. The AVIT protein family

    PubMed Central

    Kaser, Alexandra; Winklmayr, Martina; Lepperdinger, Günther; Kreil, Günther

    2003-01-01

    Homologues of a protein originally isolated from snake venom and frog skin secretions are present in many vertebrate species. They contain 80–90 amino acids, 10 of which are cysteines with identical spacing. Various names have been given to these proteins, such as mamba intestinal protein 1 (MIT1), Bv8 (Bombina variegata molecular mass ∼8 kDa), prokineticins and endocrine-gland vascular endothelial growth factor (EG-VEGF). Their amino-terminal sequences are identical, and so we propose that the sequence of their first four residues, AVIT, is used as a name for this family. From a comparison of the sequences, two types of AVIT proteins can be discerned. These proteins seem to be distributed widely in mammalian tissues and are known to bind to G-protein-coupled receptors. Members of this family have been shown to stimulate contraction of the guinea pig ileum, to cause hyperalgesia after injection into rats and to be active as specific growth factors. Moreover, the messenger RNA level of one of these AVIT proteins changes rhythmically in the region of the brain known as the suprachiasmatic nucleus. This shows that members of this new family of small proteins are involved in diverse biological processes. PMID:12728244

  9. Protein Kinases and Addiction

    PubMed Central

    Lee, Anna M.; Messing, Robert O.

    2011-01-01

    Although drugs of abuse have different chemical structures and interact with different protein targets, all appear to usurp common neuronal systems that regulate reward and motivation. Addiction is a complex disease that is thought to involve drug-induced changes in synaptic plasticity due to alterations in cell signaling, gene transcription, and protein synthesis. Recent evidence suggests that drugs of abuse interact with and change a common network of signaling pathways that include a subset of specific protein kinases. The best studied of these kinases are reviewed here and include extracellular signal-regulated kinase, cAMP-dependent protein kinase, cyclin-dependent protein kinase 5, protein kinase C, calcium/calmodulin-dependent protein kinase II, and Fyn tyrosine kinase. These kinases have been implicated in various aspects of drug addiction including acute drug effects, drug self-administration, withdrawal, reinforcement, sensitization, and tolerance. Identifying protein kinase substrates and signaling pathways that contribute to the addicted state may provide novel approaches for new pharma-cotherapies to treat drug addiction. PMID:18991950

  10. 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

  11. 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

  12. 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.

  13. 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

  14. Protein sequence databases.

    PubMed

    Apweiler, Rolf; Bairoch, Amos; Wu, Cathy H

    2004-02-01

    A variety of protein sequence databases exist, ranging from simple sequence repositories, which store data with little or no manual intervention in the creation of the records, to expertly curated universal databases that cover all species and in which the original sequence data are enhanced by the manual addition of further information in each sequence record. As the focus of researchers moves from the genome to the proteins encoded by it, these databases will play an even more important role as central comprehensive resources of protein information. Several the leading protein sequence databases are discussed here, with special emphasis on the databases now provided by the Universal Protein Knowledgebase (UniProt) consortium. PMID:15036160

  15. 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

  16. 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.

  17. The centrality of cancer proteins in human protein-protein interaction network: a revisit.

    PubMed

    Xiong, Wei; Xie, Luyu; Zhou, Shuigeng; Liu, Hui; Guan, Jihong

    2014-01-01

    Topological analysis of protein-protein interaction (PPI) networks has been widely applied to the investigation on cancer mechanisms. However, there is still a debate on whether cancer proteins exhibit more topological centrality compared to the other proteins in the human PPI network. To resolve this debate, we first identified four sets of human proteins, and then mapped these proteins into the yeast PPI network by homologous genes. Finally, we compared these proteins' properties in human and yeast PPI networks. Experiments over two real datasets demonstrated that cancer proteins tend to have higher degree and smaller clustering coefficient than non-cancer proteins. Experimental results also validated that cancer proteins have larger betweenness centrality compared to the other proteins on the STRING dataset. However, on the BioGRID dataset, the average betweenness centrality of cancer proteins is larger than that of disease and control proteins, but smaller than that of essential proteins. PMID:24878726

  18. Protein Regulation in Signal Transduction.

    PubMed

    Lee, Michael J; Yaffe, Michael B

    2016-01-01

    SUMMARYCells must respond to a diverse, complex, and ever-changing mix of signals, using a fairly limited set of parts. Changes in protein level, protein localization, protein activity, and protein-protein interactions are critical aspects of signal transduction, allowing cells to respond highly specifically to a nearly limitless set of cues and also to vary the sensitivity, duration, and dynamics of the response. Signal-dependent changes in levels of gene expression and protein synthesis play an important role in regulation of protein levels, whereas posttranslational modifications of proteins regulate their degradation, localization, and functional interactions. Protein ubiquitylation, for example, can direct proteins to the proteasome for degradation or provide a signal that regulates their interactions and/or location within the cell. Similarly, protein phosphorylation by specific kinases is a key mechanism for augmenting protein activity and relaying signals to other proteins that possess domains that recognize the phosphorylated residues. PMID:27252361

  19. 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

  20. 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

  1. PSC: protein surface classification

    PubMed Central

    Tseng, Yan Yuan; Li, Wen-Hsiung

    2012-01-01

    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. PMID:22669905

  2. 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.

  3. (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.

  4. 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)

  5. 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.

  6. 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.

  7. Emerging fluorescent protein technologies.

    PubMed

    Enterina, Jhon Ralph; Wu, Lanshi; Campbell, Robert E

    2015-08-01

    Fluorescent proteins (FPs), such as the Aequorea jellyfish green FP (GFP), are firmly established as fundamental tools that enable a wide variety of biological studies. Specifically, FPs can serve as versatile genetically encoded markers for tracking proteins, organelles, or whole cells, and as the basis for construction of biosensors that can be used to visualize a growing array of biochemical events in cells and tissues. In this review we will focus on emerging applications of FPs that represent unprecedented new directions for the field. These emerging applications include new strategies for using FPs in biosensing applications, and innovative ways of using FPs to manipulate protein function or gene expression. PMID:26043278

  8. 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

  9. 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.

  10. 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.

  11. Evolution of proteins.

    NASA Technical Reports Server (NTRS)

    Dayhoff, M. O.

    1971-01-01

    The amino acid sequences of proteins from living organisms are dealt with. The structure of proteins is first discussed; the variation in this structure from one biological group to another is illustrated by the first halves of the sequences of cytochrome c, and a phylogenetic tree is derived from the cytochrome c data. The relative geological times associated with the events of this tree are discussed. Errors which occur in the duplication of cells during the evolutionary process are examined. Particular attention is given to evolution of mutant proteins, globins, ferredoxin, and transfer ribonucleic acids (tRNA's). Finally, a general outline of biological evolution is presented.

  12. 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

  13. A Bayesian Estimator of Protein-Protein Association Probabilities

    SciTech Connect

    Gilmore, Jason M.; Auberry, Deanna L.; Sharp, Julia L.; White, Amanda M.; Anderson, Kevin K.; Daly, Don S.

    2008-07-01

    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 pull-down LC-MS assay experiments. BEPro3 is open source software that runs on both Windows XP and Mac OS 10.4 or newer versions, and is freely available from http://www.pnl.gov/statistics/BEPro3.

  14. Interactive protein manipulation

    SciTech Connect

    SNCrivelli@lbl.gov

    2003-07-01

    We describe an interactive visualization and modeling program for the creation of protein structures ''from scratch''. The input to our program is an amino acid sequence -decoded from a gene- and a sequence of predicted secondary structure types for each amino acid-provided by external structure prediction programs. Our program can be used in the set-up phase of a protein structure prediction process; the structures created with it serve as input for a subsequent global internal energy minimization, or another method of protein structure prediction. Our program supports basic visualization methods for protein structures, interactive manipulation based on inverse kinematics, and visualization guides to aid a user in creating ''good'' initial structures.

  15. 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.

  16. Engineered Proteins for Bioelectrochemistry

    NASA Astrophysics Data System (ADS)

    Akram, Muhammad Safwan; Rehman, Jawad Ur; Hall, Elizabeth A. H.

    2014-06-01

    It is only in the past two decades that excellent protein engineering tools have begun to meet parallel advances in materials chemistry, nanofabrication, and electronics. This is revealing scenarios from which synthetic enzymes can emerge, which were previously impossible, as well as interfaces with novel electrode materials. That means the control of the protein structure, electron transport pathway, and electrode surface can usher us into a new era of bioelectrochemistry. This article reviews the principle of electron transfer (ET) and considers how its application at the electrode, within the protein, and at a redox group is directing key advances in the understanding of protein structure to create systems that exhibit better efficiency and unique bioelectrochemistry.

  17. Protein Model Database

    SciTech Connect

    Fidelis, K; Adzhubej, A; Kryshtafovych, A; Daniluk, P

    2005-02-23

    The phenomenal success of the genome sequencing projects reveals the power of completeness in revolutionizing biological science. Currently it is possible to sequence entire organisms at a time, allowing for a systemic rather than fractional view of their organization and the various genome-encoded functions. There is an international plan to move towards a similar goal in the area of protein structure. This will not be achieved by experiment alone, but rather by a combination of efforts in crystallography, NMR spectroscopy, and computational modeling. Only a small fraction of structures are expected to be identified experimentally, the remainder to be modeled. Presently there is no organized infrastructure to critically evaluate and present these data to the biological community. The goal of the Protein Model Database project is to create such infrastructure, including (1) public database of theoretically derived protein structures; (2) reliable annotation of protein model quality, (3) novel structure analysis tools, and (4) access to the highest quality modeling techniques available.

  18. The Pentapeptide Repeat Proteins

    SciTech Connect

    Vetting,M.; Hegde, S.; Fajardo, J.; Fiser, A.; Roderick, S.; Takiff, H.; Blanchard, J.

    2006-01-01

    The Pentapeptide Repeat Protein (PRP) family has over 500 members in the prokaryotic and eukaryotic kingdoms. These proteins are composed of, or contain domains composed of, tandemly repeated amino acid sequences with a consensus sequence of [S, T,A, V][D, N][L, F]-[S, T,R][G]. The biochemical function of the vast majority of PRP family members is unknown. The three-dimensional structure of the first member of the PRP family was determined for the fluoroquinolone resistance protein (MfpA) from Mycobacterium tuberculosis. The structure revealed that the pentapeptide repeats encode the folding of a novel right-handed quadrilateral {beta}-helix. MfpA binds to DNA gyrase and inhibits its activity. The rod-shaped, dimeric protein exhibits remarkable size, shape and electrostatic similarity to DNA.

  19. Untying knots in proteins.

    PubMed

    Sułkowska, Joanna I; Sułkowski, Piotr; Szymczak, Piotr; Cieplak, Marek

    2010-10-13

    A shoelace can be readily untied by pulling its ends rather than its loops. Attempting to untie a native knot in a protein can also succeed or fail depending on where one pulls. However, thermal fluctuations induced by the surrounding water affect conformations stochastically and may add to the uncertainty of the outcome. When the protein is pulled by the termini, the knot can only get tightened, and any attempt at untying results in failure. We show that, by pulling specific amino acids, one may easily retract a terminal segment of the backbone from the knotting loop and untangle the knot. At still other amino acids, the outcome of pulling can go either way. We study the dependence of the untying probability on the way the protein is grasped, the pulling speed, and the temperature. Elucidation of the mechanisms underlying this dependence is critical for a successful experimental realization of protein knot untying. PMID:20857930

  20. Membrane Protein Prediction Methods

    PubMed Central

    Punta, Marco; Forrest, Lucy R.; Bigelow, Henry; Kernytsky, Andrew; Liu, Jinfeng; Rost, Burkhard

    2007-01-01

    We survey computational approaches that tackle membrane protein structure and function prediction. While describing the main ideas that have led to the development of the most relevant and novel methods, we also discuss pitfalls, provide practical hints and highlight the challenges that remain. The methods covered include: sequence alignment, motif search, functional residue identification, transmembrane segment and protein topology predictions, homology and ab initio modeling. Overall, predictions of functional and structural features of membrane proteins are improving, although progress is hampered by the limited amount of high-resolution experimental information available. While predictions of transmembrane segments and protein topology rank among the most accurate methods in computational biology, more attention and effort will be required in the future to ameliorate database search, homology and ab initio modeling. PMID:17367718

  1. 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 ...

  2. Protein Nitrogen Determination

    NASA Astrophysics Data System (ADS)

    Nielsen, S. Suzanne

    The protein content of foods can be determined by numerous methods. The Kjeldahl method and the nitrogen combustion (Dumas) method for protein analysis are based on nitrogen determination. Both methods are official for the purposes of nutrition labeling of foods. While the Kjeldahl method has been used widely for over a hundred years, the recent availability of automated instrumentation for the Dumas method in many cases is replacing use of the Kjeldahl method.

  3. 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

  4. 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.

  5. 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).

  6. 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.

  7. Stretching to Understand Proteins

    NASA Astrophysics Data System (ADS)

    Cieplak, Marek

    2007-03-01

    Mechanical stretching of single proteins has been studied experimentally for about 50 proteins yielding a variety of force patterns and values of the peak forces. We have performed a theoretical survey of 7749 proteins of known native structure and map out the landscape of possible dynamical behaviors unders stretching at constant speed. The model used is constructed based on the native geometry. It is solved by methods of molecular dynamics and validated by comparing the theoretical predictions to experimental results. We characterize the distribution of peak forces and on correlations with the system size and with the structure classification as characterized by the CATH scheme. We identify proteins with the biggest forces and show that they belong to few topology classes. We determine which protein segments act as mechanical clamps and show that, in most cases, they correspond to long stretches of parallel beta-strands, but other mechanisms are also possible. We then consider stretching by fluid flows. We show that unfolding induced by a uniform flow shows a richer behavior than that in the force clamp. The dynamics of unfolding is found to depend strongly on the selection of the amino acid, usually one of the termini, which is anchored. These features offer potentially wider diagnostic tools to investigate structure of proteins compared to experiments based on the atomic force microscopy.

  8. 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

  9. 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

  10. 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.

  11. 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

  12. 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

  13. 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.

  14. 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.

  15. 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

  16. Solid State NMR and Protein-Protein Interactions in Membranes

    PubMed Central

    Miao, Yimin; Cross, Timothy A.

    2013-01-01

    Solid state NMR spectroscopy has evolved rapidly in recent years into an excellent tool for the characterization of membrane proteins and their complexes. In the past few years it has also become clear that the structure of membrane proteins, especially helical membrane proteins is determined, in part, by the membrane environment. Therefore, the modeling of this environment by a liquid crystalline lipid bilayer for solid state NMR has generated a unique tool for the characterization of native conformational states, local and global dynamics, and high resolution structure for these proteins. Protein-protein interactions can also benefit from this solid state NMR capability to characterize membrane proteins in a native-like environment. These complexes take the form of oligomeric structures and hetero-protein interactions both with water soluble proteins and other membrane proteins. PMID:24034903

  17. Solid state NMR and protein-protein interactions in membranes.

    PubMed

    Miao, Yimin; Cross, Timothy A

    2013-12-01

    Solid state NMR spectroscopy has evolved rapidly in recent years into an excellent tool for the characterization of membrane proteins and their complexes. In the past few years it has also become clear that the structure of membrane proteins, especially helical membrane proteins is determined, in part, by the membrane environment. Therefore, the modeling of this environment by a liquid crystalline lipid bilayer for solid state NMR has generated a unique tool for the characterization of native conformational states, local and global dynamics, and high-resolution structure for these proteins. Protein-protein interactions can also benefit from this solid state NMR capability to characterize membrane proteins in a native-like environment. These complexes take the form of oligomeric structures and hetero-protein interactions both with water-soluble proteins and other membrane proteins. PMID:24034903

  18. 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

  19. Histophilus somni Surface Proteins.

    PubMed

    Corbeil, Lynette B

    2016-01-01

    The pathogen surface is usually the first site of interaction with the host. Histophilus somni was earlier thought to only have an outer membrane on its surface. Now it is known that the surface is composed of many virulence factors, including outer membrane proteins, lipooligosaccharide or endotoxin, a fibrillar network, and an exopolysaccharide. Outer membrane blebs, endotoxin, the fibrillar network, and the exopolysaccharide are also shed from the surface. This review will focus on the surface proteins of this pathogen that may colonize the mucosal surface of ruminants as a commensal or may cause pneumonia, septicemia, myocarditis, thrombotic meningoencephalitis, arthritis, and/or abortion. The major outer membrane protein has been well studied. Since its size and epitopes vary from strain to strain, it may be useful for typing strains. Iron-regulated OMPs have also received much attention because of their role in iron uptake for in vivo growth of H. somni. Other OMPs may be protective, based on passive immunization with monospecific antibodies and active immunization experiments. The surface and shed fibrillar network has been shown to be an immunoglobulin-binding protein in that it binds bovine IgG2 by the Fc portion. Two repeat domains (DR1 and DR2) have cytotoxic Fic motifs. Vaccine studies with recombinant DR2 are promising. Studies of the bacterial genome as well as comparison of surface proteins of different strains from the various H. somni syndromes and carrier states will be discussed and have provided much insight into pathogenesis and protection. PMID:26728061

  20. 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

  1. How Many Protein-Protein Interactions Types Exist in Nature?

    PubMed Central

    Mitra, Pralay; Zhang, Yang

    2012-01-01

    Protein quaternary structure universe” refers to the ensemble of all protein-protein complexes across all organisms in nature. The number of quaternary folds thus corresponds to the number of ways proteins physically interact with other proteins. This study focuses on answering two basic questions: Whether the number of protein-protein interactions is limited and, if yes, how many different quaternary folds exist in nature. By all-to-all sequence and structure comparisons, we grouped the protein complexes in the protein data bank (PDB) into 3,629 families and 1,761 folds. A statistical model was introduced to obtain the quantitative relation between the numbers of quaternary families and quaternary folds in nature. The total number of possible protein-protein interactions was estimated around 4,000, which indicates that the current protein repository contains only 42% of quaternary folds in nature and a full coverage needs approximately a quarter century of experimental effort. The results have important implications to the protein complex structural modeling and the structure genomics of protein-protein interactions. PMID:22719985

  2. How many protein-protein interactions types exist in nature?

    PubMed

    Garma, Leonardo; Mukherjee, Srayanta; Mitra, Pralay; Zhang, Yang

    2012-01-01

    "Protein quaternary structure universe" refers to the ensemble of all protein-protein complexes across all organisms in nature. The number of quaternary folds thus corresponds to the number of ways proteins physically interact with other proteins. This study focuses on answering two basic questions: Whether the number of protein-protein interactions is limited and, if yes, how many different quaternary folds exist in nature. By all-to-all sequence and structure comparisons, we grouped the protein complexes in the protein data bank (PDB) into 3,629 families and 1,761 folds. A statistical model was introduced to obtain the quantitative relation between the numbers of quaternary families and quaternary folds in nature. The total number of possible protein-protein interactions was estimated around 4,000, which indicates that the current protein repository contains only 42% of quaternary folds in nature and a full coverage needs approximately a quarter century of experimental effort. The results have important implications to the protein complex structural modeling and the structure genomics of protein-protein interactions. PMID:22719985

  3. Computational drug design targeting protein-protein interactions.

    PubMed

    Bienstock, Rachelle J

    2012-01-01

    Novel discoveries in molecular disease pathways within the cell, combined with increasing information regarding protein binding partners has lead to a new approach in drug discovery. There is interest in designing drugs to modulate protein-protein interactions as opposed to solely targeting the catalytic active site within a single enzyme or protein. There are many challenges in this new approach to drug discovery, particularly since the protein-protein interface has a larger surface area, can comprise a discontinuous epitope, and is more amorphous and less well defined than the typical drug design target, a small contained enzyme-binding pocket. Computational methods to predict modes of protein-protein interaction, as well as protein interface hot spots, have garnered significant interest, in order to facilitate the development of drugs to successfully disrupt and inhibit protein-protein interactions. This review summarizes some current methods available for computational protein-protein docking, as well as tabulating some examples of the successful design of antagonists and small molecule inhibitors for protein-protein interactions. Several of these drugs are now beginning to appear in the clinic. PMID:22316151

  4. 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

  5. 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.

  6. Collapse transition in proteins.

    PubMed

    Ziv, Guy; Thirumalai, D; Haran, Gilad

    2009-01-01

    The coil-globule transition, a tenet of the physics of polymers, has been identified in recent years as an important unresolved aspect of the initial stages of the folding of proteins. We describe the basics of the collapse transition, starting with homopolymers and continuing with proteins. Studies of denatured-state collapse under equilibrium are then presented. An emphasis is placed on single-molecule fluorescence experiments, which are particularly useful for measuring properties of the denatured state even under conditions of coexistence with the folded state. Attempts to understand the dynamics of collapse, both theoretically and experimentally, are then described. Only an upper limit for the rate of collapse has been obtained so far. Improvements in experimental and theoretical methodology are likely to continue to push our understanding of the importance of the denatured-state thermodynamics and dynamics for protein folding in the coming years. PMID:19081910

  7. Polarizable protein packing.

    PubMed

    Ng, Albert H; Snow, Christopher D

    2011-05-01

    To incorporate protein polarization effects within a protein combinatorial optimization framework, we decompose the polarizable force field AMOEBA into low order terms. Including terms up to the third-order provides a fair approximation to the full energy while maintaining tractability. We represent the polarizable packing problem for protein G as a hypergraph and solve for optimal rotamers with the FASTER combinatorial optimization algorithm. These approximate energy models can be improved to high accuracy [root mean square deviation (rmsd) < 1 kJ mol(-1)] via ridge regression. The resulting trained approximations are used to efficiently identify new, low-energy solutions. The approach is general and should allow combinatorial optimization of other many-body problems. PMID:21264879

  8. Electron transfer in proteins.

    PubMed

    Gray, H B; Winkler, J R

    1996-01-01

    Electron-transfer (ET) reactions are key steps in a diverse array of biological transformations ranging from photosynthesis to aerobic respiration. A powerful theoretical formalism has been developed that describes ET rates in terms of two parameters: the nuclear reorganization energy (lambda) and the electronic-coupling strength (HAB). Studies of ET reactions in ruthenium-modified proteins have probed lambda and HAB in several metalloproteins (cytochrome c, myoglobin, azurin). This work has shown that protein reorganization energies are sensitive to the medium surrounding the redox sites and that an aqueous environment, in particular, leads to large reorganization energies. Analyses of electronic-coupling strengths suggest that the efficiency of long-range ET depends on the protein secondary structure: beta sheets appear to mediate coupling more efficiently than alpha-helical structures, and hydrogen bonds play a critical role in both. PMID:8811189

  9. 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

  10. 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.

  11. New MAPS for misfolded proteins.

    PubMed

    Volkmar, Norbert; Fenech, Emma; Christianson, John C

    2016-06-28

    Clearing misfolded proteins from the cytoplasm is essential to maintain cellular homeostasis. Now, a parallel clearance system is described that uses the deubiquitylase USP19 to enable secretion of misfolded cytoplasmic proteins when conventional proteasomal degradation is compromised. Misfolding-associated protein secretion (MAPS) has important implications for protein quality control and prion-like transmission. PMID:27350445

  12. SOY PROTEIN NANOPARTICLES AND NANOCOMPOSITES

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Soy protein isolate (SPI) is obtained from soybean by removing soybean oil and soy carbohydrates. SPI contains more than 90% protein. Structurally, SPI is a globular protein and its aggregates in water consist of sphere-like protein particles. The number average aggregate size of SPI at pH=5.2 is...

  13. 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...

  14. Bioinformatics and Moonlighting Proteins

    PubMed Central

    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

  15. 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

  16. Epistasis in protein evolution.

    PubMed

    Starr, Tyler N; Thornton, Joseph W

    2016-07-01

    The structure, function, and evolution of proteins depend on physical and genetic interactions among amino acids. Recent studies have used new strategies to explore the prevalence, biochemical mechanisms, and evolutionary implications of these interactions-called epistasis-within proteins. Here we describe an emerging picture of pervasive epistasis in which the physical and biological effects of mutations change over the course of evolution in a lineage-specific fashion. Epistasis can restrict the trajectories available to an evolving protein or open new paths to sequences and functions that would otherwise have been inaccessible. We describe two broad classes of epistatic interactions, which arise from different physical mechanisms and have different effects on evolutionary processes. Specific epistasis-in which one mutation influences the phenotypic effect of few other mutations-is caused by direct and indirect physical interactions between mutations, which nonadditively change the protein's physical properties, such as conformation, stability, or affinity for ligands. In contrast, nonspecific epistasis describes mutations that modify the effect of many others; these typically behave additively with respect to the physical properties of a protein but exhibit epistasis because of a nonlinear relationship between the physical properties and their biological effects, such as function or fitness. Both types of interaction are rampant, but specific epistasis has stronger effects on the rate and outcomes of evolution, because it imposes stricter constraints and modulates evolutionary potential more dramatically; it therefore makes evolution more contingent on low-probability historical events and leaves stronger marks on the sequences, structures, and functions of protein families. PMID:26833806

  17. Single-cell proteins

    SciTech Connect

    Litchfield, J.H.

    1983-02-11

    Both photosynthetic and nonphotosynthetic microorganisms, grown on various carbon and energy sources, are used in fermentation processes for the production of single-cell proteins. Commercial-scale production has been limited to two algal processes, one bacterial process, and several yeast and fungal processes. High capital and operating costs and the need for extensive nutritional and toxicological assessments have limited the development and commercialization of new processes. Any increase in commercial-scale production appears to be limited to those regions of the world where low-cost carbon and energy sources are available and conventional animal feedstuff proteins, such as soybean meal or fish meal, are in short supply. (Refs. 59).

  18. Protein-based ferrogels.

    PubMed

    Mody, Puja; Hart, Cassidy; Romano, Siena; El-Magbri, Mariam; Esson, Moira M; Ibeh, Trisha; Knowlton, Elizabeth D; Zhang, Ming; Wagner, Michael J; Hartings, Matthew R

    2016-06-01

    We present a novel synthesis in which hemoglobin and Fe(2+) react, in the presence of KNO3 and KOH, to produce protein microgels that contain magnetic iron oxide nanoparticles. The synthesis results in microgels with polymer properties (denaturing and glass transition temperatures) that are consistent with the dried protein. The iron oxide nanoparticles that exhibit an average diameter of 22nm, are ferrimagnetic, and display properties consistent with Fe3O4. The multiple functional capabilities displayed by these materials: biocompatibility, magnetism, dye uptake and controlled release, and other properties archetypal of hydrogels, will make the magnetic hydrogels attractive for a number of biomedical applications. PMID:26901627

  19. 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

  20. Congenital protein hypoglycosylation diseases

    PubMed Central

    Sparks, Susan E

    2012-01-01

    Glycosylation is an essential process by which sugars are attached to proteins and lipids. Complete lack of glycosylation is not compatible with life. Because of the widespread function of glycosylation, inherited disorders of glycosylation are multisystemic. Since the identification of the first defect on N-linked glycosylation in the 1980s, there are over 40 different congenital protein hypoglycosylation diseases. This review will include defects of N-linked glycosylation, O-linked glycosylation and disorders of combined N- and O-linked glycosylation. PMID:23776380

  1. 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

  2. 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

  3. Conformation Distributions in Adsorbed Proteins.

    NASA Astrophysics Data System (ADS)

    Meuse, Curtis W.; Hubbard, Joseph B.; Vrettos, John S.; Smith, Jackson R.; Cicerone, Marcus T.

    2007-03-01

    While the structural basis of protein function is well understood in the biopharmaceutical and biotechnology industries, few methods for the characterization and comparison of protein conformation distributions are available. New methods capable of measuring the stability of protein conformations and the integrity of protein-protein, protein-ligand and protein-surface interactions both in solution and on surfaces are needed to help the development of protein-based products. We are developing infrared spectroscopy methods for the characterization and comparison of molecular conformation distributions in monolayers and in solutions. We have extracted an order parameter describing the orientational and conformational variations of protein functional groups around the average molecular values from a single polarized spectrum. We will discuss the development of these methods and compare them to amide hydrogen/deuterium exchange methods for albumin in solution and on different polymer surfaces to show that our order parameter is related to protein stability.

  4. Extreme multifunctional proteins identified from a human protein interaction network

    PubMed Central

    Chapple, Charles E.; Robisson, Benoit; Spinelli, Lionel; Guien, Céline; Becker, Emmanuelle; Brun, Christine

    2015-01-01

    Moonlighting proteins are a subclass of multifunctional proteins whose functions are unrelated. Although they may play important roles in cells, there has been no large-scale method to identify them, nor any effort to characterize them as a group. Here, we propose the first method for the identification of ‘extreme multifunctional' proteins from an interactome as a first step to characterize moonlighting proteins. By combining network topological information with protein annotations, we identify 430 extreme multifunctional proteins (3% of the human interactome). We show that the candidates form a distinct sub-group of proteins, characterized by specific features, which form a signature of extreme multifunctionality. Overall, extreme multifunctional proteins are enriched in linear motifs and less intrinsically disordered than network hubs. We also provide MoonDB, a database containing information on all the candidates identified in the analysis and a set of manually curated human moonlighting proteins. PMID:26054620

  5. 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

  6. 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.

  7. Transient protein-protein interactions visualized by solution NMR.

    PubMed

    Liu, Zhu; Gong, Zhou; Dong, Xu; Tang, Chun

    2016-01-01

    Proteins interact with each other to establish their identities in cell. The affinities for the interactions span more than ten orders of magnitude, and KD values in μM-mM regimen are considered transient and are important in cell signaling. Solution NMR including diamagnetic and paramagnetic techniques has enabled atomic-resolution depictions of transient protein-protein interactions. Diamagnetic NMR allows characterization of protein complexes with KD values up to several mM, whereas ultraweak and fleeting complexes can be modeled with the use of paramagnetic NMR especially paramagnetic relaxation enhancement (PRE). When tackling ever-larger protein complexes, PRE can be particularly useful in providing long-range intermolecular distance restraints. As NMR measurements are averaged over the ensemble of complex structures, structural information for dynamic protein-protein interactions besides the stereospecific one can often be extracted. Herein the protein interaction dynamics are exemplified by encounter complexes, alternative binding modes, and coupled binding/folding of intrinsically disordered proteins. Further integration of NMR with other biophysical techniques should allow better visualization of transient protein-protein interactions. In particular, single-molecule data may facilitate the interpretation of ensemble-averaged NMR data. Though same structures of proteins and protein complexes were found in cell as in diluted solution, we anticipate that the dynamics of transient protein protein-protein interactions be different, which awaits awaits exploration by NMR. This article is part of a Special Issue entitled: Physiological Enzymology and Protein Functions. This article is part of a Special Issue entitled: Physiological Enzymology and Protein Functions. PMID:25896389

  8. 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.

  9. 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.

  10. Cosolvent assisted protein refolding.

    PubMed

    Cleland, J L; Wang, D I

    1990-12-01

    The use of cosolvents in aqueous systems has been shown to enhance protein refolding and decrease aggregation. In this study, we have used polyethylene glycol (PEG) in the molecular weight range of 1000 to 8000 Daltons to effectively increase the rate of refolding and prevent aggregation of the model protein, bovine carbonic anhydrase B (CAB). At concentrations of 3 and 30 g/l, PEG increased the rate of recovery of active protein in the absence of aggregation. Using 3 g/l PEG (3350 MW), the refolding rate was three fold greater than the observed normal refolding rate. The observed rate enhancement was caused by PEG acting on the first intermediate in the CAB refolding pathway to increase the rate of formation of the second intermediate. The interaction of PEG with the first intermediate also prevented its self-association during refolding and at equilibrium. The stabilization of this first intermediate resulted in complete recovery of active protein under normal aggregating conditions. PMID:1367488

  11. 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. PMID:26387108

  12. 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

  13. 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.

  14. Protein-protein and protein-salt interactions in aqueous protein solutions containing concentrated electrolytes

    SciTech Connect

    Curtis, R.A.; Blanch, H.W.; Prausnitz, J.M.

    1998-01-05

    Protein-protein and protein-salt interactions have been obtained for ovalbumin in solutions of ammonium sulfate and for lysozyme in solutions of ammonium sulfate, sodium chloride, potassium isothiocyanate, and potassium chloride. The two-body interactions between ovalbumin molecules in concentrated ammonium-sulfate solutions can be described by the DLVO potentials plus a potential that accounts for the decrease in free volume available to the protein due to the presence of the salt ions. The interaction between ovalbumin and ammonium sulfate is unfavorable, reflecting the kosmotropic nature of sulfate anions. Lysozyme-lysozyme interactions cannot be described by the above potentials because anion binding to lysozyme alters these interactions. Lysozyme-isothiocyanate complexes are strongly attractive due to electrostatic interactions resulting from bridging by the isothiocyanate ion. Lysozyme-lysozyme interactions in sulfate solutions are more repulsive than expected, possibly resulting from a larger excluded volume of a lysozyme-sulfate bound complex or perhaps, hydration forces between the lysozyme-sulfate complexes.

  15. 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.

  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. 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

  18. 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

  19. 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

  20. Thermal unfolding of proteins

    NASA Astrophysics Data System (ADS)

    Cieplak, Marek; Sułkowska, Joanna I.

    2005-11-01

    Thermal unfolding of proteins is compared to folding and mechanical stretching in a simple topology-based dynamical model. We define the unfolding time and demonstrate its low-temperature divergence. Below a characteristic temperature, contacts break at separate time scales and unfolding proceeds approximately in a way reverse to folding. Features in these scenarios agree with experiments and atomic simulations on titin.

  1. 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.

  2. Protein denaturing on Nanospheres

    NASA Astrophysics Data System (ADS)

    Forrest, James; Teichroeb, Jonathan

    2007-03-01

    We have used localized surface plasmon resonance (LSPR) to monitor the structural changes that accompany thermal denaturing of Bovine Serum Albumin(BSA) adsorbed onto gold nanospheres of size 5nm-60nm. The effect of the protein on the LSPR was monitored by visible extinction spectroscopy. The position of the resonance is affected by the conformation of the adsorbed protein layer, and as such can be used as a very sensitive probe of thermal denaturing that is specific to the adsorbed protein. The results are compared to detailed calculations and show that full calculations can lead to significant increases in knowledge where gold nanospheres are used as biosensors. Thermal denaturing on spheres with diameter > 20 nm show strong similarity to bulk calorimetric studies of BSA in solution. BSA adsorbed on nanospheres with d<= 15 nm shows a qualitative difference in behavior, suggesting a sensitivity of denaturing characteristics on local surface curvature. Studies of isothermal denaturing kinetics were used to obtain an activatiuon barrier for thermal denaturing. This activation barrier also exhibited a strong dependence on nanoparticle size. These results may have important implications for other protein-nanoparticle interactions.

  3. [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

  4. The Protein Data Bank

    PubMed Central

    Berman, Helen M.; Westbrook, John; Feng, Zukang; Gilliland, Gary; Bhat, T. N.; Weissig, Helge; Shindyalov, Ilya N.; Bourne, Philip E.

    2000-01-01

    The Protein Data Bank (PDB; http://www.rcsb.org/pdb/ ) is the single worldwide archive of structural data of biological macromolecules. This paper describes the goals of the PDB, the systems in place for data deposition and access, how to obtain further information, and near-term plans for the future development of the resource. PMID:10592235

  5. 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

  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. Perspectives on protein crystallisation

    SciTech Connect

    Ochi, T.; Stojanoff, V.; Bolanos-Garcia, V.M.; Moreno, A.

    2009-12-11

    This final part on 'perspectives' is focused on new strategies that can be used to crystallise proteins and improve the crystal quality of macromolecular complexes using any of the methods reviewed in this focused issue. Some advantages and disadvantages, limitations, and plausible applications to high-resolution X-ray crystallography are discussed.

  8. Bayesian Estimator of Protein-Protein Association Probabilities

    Energy Science and Technology Software Center (ESTSC)

    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.

  9. Homeodomain proteins: an update.

    PubMed

    Bürglin, Thomas R; Affolter, Markus

    2016-06-01

    Here, we provide an update of our review on homeobox genes that we wrote together with Walter Gehring in 1994. Since then, comprehensive surveys of homeobox genes have become possible due to genome sequencing projects. Using the 103 Drosophila homeobox genes as example, we present an updated classification. In animals, there are 16 major classes, ANTP, PRD, PRD-LIKE, POU, HNF, CUT (with four subclasses: ONECUT, CUX, SATB, and CMP), LIM, ZF, CERS, PROS, SIX/SO, plus the TALE superclass with the classes IRO, MKX, TGIF, PBC, and MEIS. In plants, there are 11 major classes, i.e., HD-ZIP (with four subclasses: I to IV), WOX, NDX, PHD, PLINC, LD, DDT, SAWADEE, PINTOX, and the two TALE classes KNOX and BEL. Most of these classes encode additional domains apart from the homeodomain. Numerous insights have been obtained in the last two decades into how homeodomain proteins bind to DNA and increase their specificity by interacting with other proteins to regulate cell- and tissue-specific gene expression. Not only protein-DNA base pair contacts are important for proper target selection; recent experiments also reveal that the shape of the DNA plays a role in specificity. Using selected examples, we highlight different mechanisms of homeodomain protein-DNA interaction. The PRD class of homeobox genes was of special interest to Walter Gehring in the last two decades. The PRD class comprises six families in Bilateria, and tinkers with four different motifs, i.e., the PAIRED domain, the Groucho-interacting motif EH1 (aka Octapeptide or TN), the homeodomain, and the OAR motif. Homologs of the co-repressor protein Groucho are also present in plants (TOPLESS), where they have been shown to interact with small amphipathic motives (EAR), and in yeast (TUP1), where we find an EH1-like motif in MATα2. PMID:26464018

  10. Phosphorylation of human link proteins

    SciTech Connect

    Oester, D.A.; Caterson, B.; Schwartz, E.R.

    1986-06-13

    Three link proteins of 48, 44 and 40 kDa were purified from human articular cartilage and identified with monoclonal anti-link protein antibody 8-A-4. Two sets of lower molecular weight proteins of 30-31 kDa and 24-26 kDa also contained link protein epitopes recognized by the monoclonal antibody and were most likely degradative products of the intact link proteins. The link proteins of 48 and 40 kDa were identified as phosphoproteins while the 44 kDa link protein did not contain /sup 32/P. The phosphorylated 48 and 40 kDa link proteins contained approximately 2 moles PO/sub 4//mole link protein.

  11. 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

  12. 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.

  13. 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

  14. 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

  15. Protein-protein interactions in DNA mismatch repair.

    PubMed

    Friedhoff, Peter; Li, Pingping; Gotthardt, Julia

    2016-02-01

    The principal DNA mismatch repair proteins MutS and MutL are versatile enzymes that couple DNA mismatch or damage recognition to other cellular processes. Besides interaction with their DNA substrates this involves transient interactions with other proteins which is triggered by the DNA mismatch or damage and controlled by conformational changes. Both MutS and MutL proteins have ATPase activity, which adds another level to control their activity and interactions with DNA substrates and other proteins. Here we focus on the protein-protein interactions, protein interaction sites and the different levels of structural knowledge about the protein complexes formed with MutS and MutL during the mismatch repair reaction. PMID:26725162

  16. 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.

  17. Affinity purification of proteins binding to GST fusion proteins.

    PubMed

    Swaffield, J C; Johnston, S A

    2001-05-01

    This unit describes the use of proteins fused to glutathione-S-transferase (GST fusion proteins) to affinity purify other proteins, a technique also known as GST pulldown purification. The describes a strategy in which a GST fusion protein is bound to agarose affinity beads and the complex is then used to assay the binding of a specific test protein that has been labeled with [35S]methionine by in vitro translation. However, this method can be adapted for use with other types of fusion proteins; for example, His6, biotin tags, or maltose-binding protein fusions (MBP), and these may offer particular advantages. A describes preparation of an E. coli extract that is added to the reaction mixture with purified test protein to reduce nonspecific binding. PMID:18265191

  18. 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

  19. 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.

  20. Protein identification and Peptide expression resolver: harmonizing protein identification with protein expression data.

    PubMed

    Kearney, Paul; Butler, Heather; Eng, Kevin; Hugo, Patrice

    2008-01-01

    Proteomic discovery platforms generate both peptide expression information and protein identification information. Peptide expression data are used to determine which peptides are differentially expressed between study cohorts, and then these peptides are targeted for protein identification. In this paper, we demonstrate that peptide expression information is also a powerful tool for enhancing confidence in protein identification results. Specifically, we evaluate the following hypothesis: tryptic peptides originating from the same protein have similar expression profiles across samples in the discovery study. Evidence supporting this hypothesis is provided. This hypothesis is integrated into a protein identification tool, PIPER (Protein Identification and Peptide Expression Resolver), that reduces erroneous protein identifications below 5%. PIPER's utility is illustrated by application to a 72-sample biomarker discovery study where it is demonstrated that false positive protein identifications can be reduced below 5%. Consequently, it is recommended that PIPER methodology be incorporated into proteomic studies where both protein expression and identification data are collected. PMID:18062667

  1. Predicting Disease-Related Proteins Based on Clique Backbone in Protein-Protein Interaction Network

    PubMed Central

    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. PMID:25013377

  2. 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

  3. 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

  4. A magnetic protein biocompass.

    PubMed

    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. PMID:26569474

  5. Bone Morphogenetic Proteins.

    PubMed

    Katagiri, Takenobu; Watabe, Tetsuro

    2016-01-01

    Bone morphogenetic proteins (BMPs), originally identified as osteoinductive components in extracts derived from bone, are now known to play important roles in a wide array of processes during formation and maintenance of various organs including bone, cartilage, muscle, kidney, and blood vessels. BMPs and the related "growth and differentiation factors" (GDFs) are members of the transforming growth factor β (TGF-β) family, and transduce their signals through type I and type II serine-threonine kinase receptors and their intracellular downstream effectors, including Smad proteins. Furthermore, BMP signals are finely tuned by various agonists and antagonists. Because deregulation of the BMP activity at multiple steps in signal transduction is linked to a wide variety of human diseases, therapeutic use of activators and inhibitors of BMP signaling will provide potential avenues for the treatment of the human disorders that are caused by hypo- and hyperactivation of BMP signals, respectively. PMID:27252362

  6. Protein mediated membrane adhesion

    NASA Astrophysics Data System (ADS)

    Carlson, Andreas; Mahadevan, L.

    2015-05-01

    Adhesion in the context of mechanical attachment, signaling, and movement in cellular dynamics is mediated by the kinetic interactions between membrane-embedded proteins in an aqueous environment. Here, we present a minimal theoretical framework for the dynamics of membrane adhesion that accounts for the kinetics of protein binding, the elastic deformation of the membrane, and the hydrodynamics of squeeze flow in the membrane gap. We analyze the resulting equations using scaling estimates to characterize the spatiotemporal features of the adhesive patterning and corroborate them using numerical simulations. In addition to characterizing aspects of cellular dynamics, our results might also be applicable to a range of phenomena in physical chemistry and materials science where flow, deformation, and kinetics are coupled to each other in slender geometries.

  7. Electron Flow through Proteins

    PubMed Central

    Gray, Harry B.; Winkler, Jay R.

    2009-01-01

    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 FeII to RuIII and CuI to RuIII 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. PMID:20161522

  8. 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.

  9. The interface of protein structure, protein biophysics, and molecular evolution

    PubMed Central

    Liberles, David A; Teichmann, Sarah A; Bahar, Ivet; Bastolla, Ugo; Bloom, Jesse; Bornberg-Bauer, Erich; Colwell, Lucy J; de Koning, A P Jason; Dokholyan, Nikolay V; Echave, Julian; Elofsson, Arne; Gerloff, Dietlind L; Goldstein, Richard A; Grahnen, Johan A; Holder, Mark T; Lakner, Clemens; Lartillot, Nicholas; Lovell, Simon C; Naylor, Gavin; Perica, Tina; Pollock, David D; Pupko, Tal; Regan, Lynne; Roger, Andrew; Rubinstein, Nimrod; Shakhnovich, Eugene; Sjölander, Kimmen; Sunyaev, Shamil; Teufel, Ashley I; Thorne, Jeffrey L; Thornton, Joseph W; Weinreich, Daniel M; Whelan, Simon

    2012-01-01

    Abstract The interface of protein structural biology, protein biophysics, molecular evolution, and molecular population genetics forms the foundations for a mechanistic understanding of many aspects of protein biochemistry. Current efforts in interdisciplinary protein modeling are in their infancy and the state-of-the art of such models is described. Beyond the relationship between amino acid substitution and static protein structure, protein function, and corresponding organismal fitness, other considerations are also discussed. More complex mutational processes such as insertion and deletion and domain rearrangements and even circular permutations should be evaluated. The role of intrinsically disordered proteins is still controversial, but may be increasingly important to consider. Protein geometry and protein dynamics as a deviation from static considerations of protein structure are also important. Protein expression level is known to be a major determinant of evolutionary rate and several considerations including selection at the mRNA level and the role of interaction specificity are discussed. Lastly, the relationship between modeling and needed high-throughput experimental data as well as experimental examination of protein evolution using ancestral sequence resurrection and in vitro biochemistry are presented, towards an aim of ultimately generating better models for biological inference and prediction. PMID:22528593

  10. Improved method for protein complex detection using bottleneck proteins

    PubMed Central

    2013-01-01

    Background Detecting protein complexes is one of essential and fundamental tasks in understanding various biological functions or processes. Therefore accurate identification of protein complexes is indispensable. Methods For more accurate detection of protein complexes, we propose an algorithm which detects dense protein sub-networks of which proteins share closely located bottleneck proteins. The proposed algorithm is capable of finding protein complexes which allow overlapping with each other. Results We applied our algorithm to several PPI (Protein-Protein Interaction) networks of Saccharomyces cerevisiae and Homo sapiens, and validated our results using public databases of protein complexes. The prediction accuracy was even more improved over our previous work which used also bottleneck information of the PPI network, but showed limitation when predicting small-sized protein complex detection. Conclusions Our algorithm resulted in overlapping protein complexes with significantly improved F1 score over existing algorithms. This result comes from high recall due to effective network search, as well as high precision due to proper use of bottleneck information during the network search. PMID:23566214

  11. Heat shock proteins: molecular chaperones of protein biogenesis.

    PubMed Central

    Craig, E A; Gambill, B D; Nelson, R J

    1993-01-01

    Heat shock proteins (Hsps) were first identified as proteins whose synthesis was enhanced by stresses such as an increase in temperature. Recently, several of the major Hsps have been shown to be intimately involved in protein biogenesis through a direct interaction with a wide variety of proteins. As a reflection of this role, these Hsps have been referred to as molecular chaperones. Hsp70s interact with incompletely folded proteins, such as nascent chains on ribosomes and proteins in the process of translocation from the cytosol into mitochondria and the endoplasmic reticulum. Hsp60 also binds to unfolded proteins, preventing aggregation and facilitating protein folding. Although less well defined, other Hsps such as Hsp90 also play important roles in modulating the activity of a number of proteins. The function of the proteolytic system is intertwined with that of molecular chaperones. Several components of this system, encoded by heat-inducible genes, are responsible for the degradation of abnormal or misfolded proteins. The budding yeast Saccharomyces cerevisiae has proven very useful in the analysis of the role of molecular chaperones in protein maturation, translocation, and degradation. In this review, results of experiments are discussed within the context of experiments with other organisms in an attempt to describe the current state of understanding of these ubiquitous and important proteins. PMID:8336673

  12. 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.

  13. 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.

  14. 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.

  15. Protein threading by learning

    PubMed Central

    Chang, Iksoo; Cieplak, Marek; Dima, Ruxandra I.; Maritan, Amos; Banavar, Jayanth R.

    2001-01-01

    By using techniques borrowed from statistical physics and neural networks, we determine the parameters, associated with a scoring function, that are chosen optimally to ensure complete success in threading tests in a training set of proteins. These parameters provide a quantitative measure of the propensities of amino acids to be buried or exposed and to be in a given secondary structure and are a good starting point for solving both the threading and design problems. PMID:11717394

  16. 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.

  17. Distortions in protein helices.

    PubMed

    Geetha, V

    1996-08-01

    alpha-helices are the most common secondary structures in observed proteins. However, they are not always found in ideal helical conformation and they often exhibit structural distortions. Quantification of these irregularities become essential in understanding the packing of helices and therefore, their role in the functional characteristics of the protein. The backbone torsions phi, psi are of limited utility in this endeavor, because distorted helices often maintain the backbone geometry. The local compensatory effects are responsible for the preservation of the entire hydrogen bond network of the helical stretch. Earlier descriptions of helical linearity and curvature rest mostly on approximation, thus motivating the search for a better method for understanding and quantifying helical irregularities. We developed a method which involves the rotation and superposition of identical repeating units of the protein by the quaternion method. The set of parameters derived from the rotation-superposition algorithm helps in identifying the bends and kinks which are not necessarily induced by unusual amino acids like proline. The quantification of irregularities of observed helices might lead to a better understanding of their packing interactions. PMID:8842770

  18. 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.

  19. Type Zero Copper Proteins

    PubMed Central

    Lancaster, Kyle M.; DeBeer George, Serena; Yokoyama, Keiko; Richards, John H.; Gray, Harry B.

    2009-01-01

    Copper proteins play key roles in biological processes such as electron transfer and dioxygen activation; the active site of each of these proteins is classified as either type 1, 2, or 3, depending on its optical and electron paramagnetic resonance properties. We have built a new type of site that we call “type zero copper” by incorporating leucine, isoleucine, or phenylalanine in place of methionine at position 121 in C112D Pseudomonas aeruginosa azurin. X-ray crystallographic analysis shows that these sites adopt distorted tetrahedral geometries, with an unusually short Cu-O(G45 carbonyl) bond (2.35–2.55 Å). Relatively weak absorption near 800 nm and narrow parallel hyperfine splittings in EPR spectra are the spectroscopic signatures of type zero copper. Copper K-edge x-ray absorption spectra suggest elevated Cu(II) 4p character in the d-electron ground state. Cyclic voltammetric experiments demonstrate that the electron transfer reactivities of type zero azurins are enhanced relative to that of the corresponding type 2 (C112D) protein. PMID:20305734

  20. 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

  1. 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.

  2. The caveolin proteins

    PubMed Central

    Williams, Terence M; Lisanti, Michael P

    2004-01-01

    The caveolin gene family has three members in vertebrates: caveolin-1, caveolin-2, and caveolin-3. So far, most caveolin-related research has been conducted in mammals, but the proteins have also been found in other animals, including Xenopus laevis, Fugu rubripes, and Caenorhabditis elegans. Caveolins can serve as protein markers of caveolae ('little caves'), invaginations in the plasma membrane 50-100 nanometers in diameter. Caveolins are found predominantly at the plasma membrane but also in the Golgi, the endoplasmic reticulum, in vesicles, and at cytosolic locations. They are expressed ubiquitously in mammals, but their expression levels vary considerably between tissues. The highest levels of caveolin-1 (also called caveolin, Cav-1 and VIP2I) are found in terminally-differentiated cell types, such as adipocytes, endothelia, smooth muscle cells, and type I pneumocytes. Caveolin-2 (Cav-2) is colocalized and coexpressed with Cav-1 and requires Cav-1 for proper membrane targeting; the Cav-2 gene also maps to the same chromosomal region as Cav-1 (7q31.1 in humans). Caveolin-3 (Cav-3) has greater protein-sequence similarity to Cav-1 than to Cav-2, but it is expressed mainly in muscle cells, including smooth, skeletal, and cardiac myocytes. Caveolins participate in many important cellular processes, including vesicular transport, cholesterol homeostasis, signal transduction, and tumor suppression. PMID:15003112

  3. 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

  4. Bioinformatics in protein analysis.

    PubMed

    Persson, B

    2000-01-01

    The chapter gives an overview of bioinformatic techniques of importance in protein analysis. These include database searches, sequence comparisons and structural predictions. Links to useful World Wide Web (WWW) pages are given in relation to each topic. Databases with biological information are reviewed with emphasis on databases for nucleotide sequences (EMBL, GenBank, DDBJ), genomes, amino acid sequences (Swissprot, PIR, TrEMBL, GenePept), and three-dimensional structures (PDB). Integrated user interfaces for databases (SRS and Entrez) are described. An introduction to databases of sequence patterns and protein families is also given (Prosite, Pfam, Blocks). Furthermore, the chapter describes the widespread methods for sequence comparisons, FASTA and BLAST, and the corresponding WWW services. The techniques involving multiple sequence alignments are also reviewed: alignment creation with the Clustal programs, phylogenetic tree calculation with the Clustal or Phylip packages and tree display using Drawtree, njplot or phylo_win. Finally, the chapter also treats the issue of structural prediction. Different methods for secondary structure predictions are described (Chou-Fasman, Garnier-Osguthorpe-Robson, Predator, PHD). Techniques for predicting membrane proteins, antigenic sites and postranslational modifications are also reviewed. PMID:10803381

  5. Protein secretion in Bacillus species.

    PubMed Central

    Simonen, M; Palva, I

    1993-01-01

    Bacilli secrete numerous proteins into the environment. Many of the secretory proteins, their export signals, and their processing steps during secretion have been characterized in detail. In contrast, the molecular mechanisms of protein secretion have been relatively poorly characterized. However, several components of the protein secretion machinery have been identified and cloned recently, which is likely to lead to rapid expansion of the knowledge of the protein secretion mechanism in Bacillus species. Comparison of the presently known export components of Bacillus species with those of Escherichia coli suggests that the mechanism of protein translocation across the cytoplasmic membrane is conserved among gram-negative and gram-positive bacteria differences are found in steps preceding and following the translocation process. Many of the secretory proteins of bacilli are produced industrially, but several problems have been encountered in the production of Bacillus heterologous secretory proteins. In the final section we discuss these problems and point out some possibilities to overcome them. PMID:8464403

  6. 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)

  7. Current Protocols in Protein Science

    PubMed Central

    Huynh, Kathy

    2015-01-01

    The purification of recombinant proteins for biochemical assays and structural studies is time-consuming and presents inherent difficulties that depend on the optimization of protein stability. The use of dyes to monitor thermal denaturation of proteins with sensitive fluorescence detection enables the rapid and inexpensive determination of protein stability using real-time PCR instruments. By screening a wide range of solution conditions and additives in 96-well format, the thermal shift assay easily identifies conditions that significantly enhance the stability of recombinant proteins. The same approach can be used as a low cost, initial screen to discover new protein:ligand interactions by capitalizing on increases in protein stability that typically occur upon ligand binding. This unit presents a methodological workflow for the small-scale, high-throughout thermal denaturation of recombinant proteins in the presence of SYPRO Orange dye. PMID:25640896

  8. 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.

  9. How do chaperonins fold protein?

    PubMed Central

    Motojima, Fumihiro

    2015-01-01

    Protein folding is a biological process that is essential for the proper functioning of proteins in all living organisms. In cells, many proteins require the assistance of molecular chaperones for their folding. Chaperonins belong to a class of molecular chaperones that have been extensively studied. However, the mechanism by which a chaperonin mediates the folding of proteins is still controversial. Denatured proteins are folded in the closed chaperonin cage, leading to the assumption that denatured proteins are completely encapsulated inside the chaperonin cage. In contrast to the assumption, we recently found that denatured protein interacts with hydrophobic residues at the subunit interfaces of the chaperonin, and partially protrude out of the cage. In this review, we will explain our recent results and introduce our model for the mechanism by which chaperonins accelerate protein folding, in view of recent findings.

  10. 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.

  11. 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.

  12. Lattice Tube Model of Proteins

    NASA Astrophysics Data System (ADS)

    Banavar, Jayanth R.; Cieplak, Marek; Maritan, Amos

    2004-11-01

    We present a new lattice model for proteins that incorporates a tubelike anisotropy by introducing a preference for mutually parallel alignments in the conformations. The model is demonstrated to capture many aspects of real proteins.

  13. Geometry and physics of proteins

    NASA Astrophysics Data System (ADS)

    Banavar, Jayanth R.; Cieplak, Marek; Hoang, Trinh X.; Maritan, Amos

    2005-03-01

    We recall some of the key lessons of protein research over the last several decades and show that they strongly suggest a new framework for understanding proteins. The unified framework is useful for understanding protein folding, amyloid formation and protein interactions and has important implications for natural selection. The experimental data and our new approach, supported by computer simulations, reveal an astonishing simplicity underlying the protein problem. REFERENCES: Banavar, J. R. and Maritan, A. (2003). Colloquium: Geometrical approach to protein folding: A tube picture. Rev. Mod. Phys. 75, 23. Banavar, J. R., Hoang, T. X., Maritan, A., Seno, F. and Trovato, A., (2004). A unified perspective on proteins -- a physics approach. Phys. Rev. E 70, 041905. Banavar, J. R., Cieplak, M. and Maritan, A., (2004). Lattice tube model of proteins, Phys. Rev. Lett. (in press).

  14. Computational Characterization of Moonlighting Proteins

    PubMed Central

    Khan, Ishita K; Kihara, Daisuke

    2016-01-01

    Moonlighting proteins perform multiple independent cellular functions within one polypeptide chain. Moonlighting proteins switch functions depending on various factors including the cell type in which they are expressed, cellular location, oligomerization status, and the binding of different ligands at different sites. Although an increasing number of moonlighting proteins have been experimentally identified in recent years, the quantity of known moonlighting proteins is insufficient to elucidate their overall landscape. Moreover, most moonlighting proteins have been identified as a serendipitous discovery. Hence, characterization of moonlighting proteins using bioinformatics approaches can have a significant impact on the overall understanding of protein function. In this work, we provide a short review of existing computational approaches for illuminating the functional diversity of moonlighting proteins. PMID:25399606

  15. 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...

  16. 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

  17. Protein corona: Opportunities and challenges.

    PubMed

    Zanganeh, Saeid; Spitler, Ryan; Erfanzadeh, Mohsen; Alkilany, Alaaldin M; Mahmoudi, Morteza

    2016-06-01

    In contact with biological fluids diverse type of biomolecules (e.g., proteins) adsorb onto nanoparticles forming protein corona. Surface properties of the coated nanoparticles, in terms of type and amount of associated proteins, dictate their interactions with biological systems and thus biological fate, therapeutic efficiency and toxicity. In this perspective, we will focus on the recent advances and pitfalls in the protein corona field. PMID:26783938

  18. Knot theory in understanding proteins.

    PubMed

    Mishra, Rama; Bhushan, Shantha

    2012-12-01

    This paper aims to enthuse mathematicians, especially topologists, knot theorists and geometers to examine problems in the study of proteins. We have highlighted those advances and breakthroughs in knot theory that directly and indirectly help in understanding proteins. We have discussed the phenomena of knotting of protein backbone. This paper also provides a few open questions for knot theorists, the answers to which will help in further understanding of proteins. PMID:22105789

  19. 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

  20. Protein Separation and Characterization Procedures

    NASA Astrophysics Data System (ADS)

    Smith, Denise M.

    Many protein separation techniques are available to food scientists. Several of the separation techniques described in this chapter are used commercially for the production of food or food ingredients, whereas others are used to purify a protein from a food for further study in the laboratory. In general, separation techniques exploit the biochemical differences in protein solubility, size, charge, adsorption characteristics, and biological affinities for other molecules. These physical characteristics then are used to purify individual proteins from complex mixtures.

  1. Protein function annotation using protein domain family resources.

    PubMed

    Das, Sayoni; Orengo, Christine A

    2016-01-15

    As a result of the genome sequencing and structural genomics initiatives, we have a wealth of protein sequence and structural data. However, only about 1% of these proteins have experimental functional annotations. As a result, computational approaches that can predict protein functions are essential in bridging this widening annotation gap. This article reviews the current approaches of protein function prediction using structure and sequence based classification of protein domain family resources with a special focus on functional families in the CATH-Gene3D resource. PMID:26434392

  2. 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

  3. Identification of essential proteins based on ranking edge-weights in protein-protein interaction networks.

    PubMed

    Wang, Yan; Sun, Huiyan; Du, Wei; Blanzieri, Enrico; Viero, Gabriella; Xu, Ying; Liang, Yanchun

    2014-01-01

    Essential proteins are those that are indispensable to cellular survival and development. Existing methods for essential protein identification generally rely on knock-out experiments and/or the relative density of their interactions (edges) with other proteins in a Protein-Protein Interaction (PPI) network. Here, we present a computational method, called EW, to first rank protein-protein interactions in terms of their Edge Weights, and then identify sub-PPI-networks consisting of only the highly-ranked edges and predict their proteins as essential proteins. We have applied this method to publicly-available PPI data on Saccharomyces cerevisiae (Yeast) and Escherichia coli (E. coli) for essential protein identification, and demonstrated that EW achieves better performance than the state-of-the-art methods in terms of the precision-recall and Jackknife measures. The highly-ranked protein-protein interactions by our prediction tend to be biologically significant in both the Yeast and E. coli PPI networks. Further analyses on systematically perturbed Yeast and E. coli PPI networks through randomly deleting edges demonstrate that the proposed method is robust and the top-ranked edges tend to be more associated with known essential proteins than the lowly-ranked edges. PMID:25268881

  4. Evolution of Chloroplast J Proteins

    PubMed Central

    Chiu, Chi-Chou; Chen, Lih-Jen; Su, Pai-Hsiang; Li, Hsou-min

    2013-01-01

    Hsp70 chaperones are involved in multiple biological processes and are recruited to specific processes by designated J domain-containing cochaperones, or J proteins. To understand the evolution and functions of chloroplast Hsp70s and J proteins, we identified the Arabidopsis chloroplast J protein constituency using a combination of genomic and proteomic database searches and individual protein import assays. We show that Arabidopsis chloroplasts have at least 19 J proteins, the highest number of confirmed J proteins for any organelle. These 19 J proteins are classified into 11 clades, for which cyanobacteria and glaucophytes only have homologs for one clade, green algae have an additional three clades, and all the other 7 clades are specific to land plants. Each clade also possesses a clade-specific novel motif that is likely used to interact with different client proteins. Gene expression analyses indicate that most land plant-specific J proteins show highly variable expression in different tissues and are down regulated by low temperatures. These results show that duplication of chloroplast Hsp70 in land plants is accompanied by more than doubling of the number of its J protein cochaperones through adding new J proteins with novel motifs, not through duplications within existing families. These new J proteins likely recruit chloroplast Hsp70 to perform tissue specific functions related to biosynthesis rather than to stress resistance. PMID:23894646

  5. Transglutaminase Polymerization of Peanut Proteins

    Technology Transfer Automated Retrieval System (TEKTRAN)

    INTRODUCTION: Transglutaminase (TGase) [protein-glutamine:amine gamma-glutamyl-transferase, EC 2.3.2.13]promotes protein cross-linking reactions through an acyl transferase mechanism involving protein-bound glutaminyl residues and primary amines (1), including the epsilon-amino group of lysine resid...

  6. 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

  7. 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

  8. 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).

  9. 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.

  10. Current Experimental Methods for Characterizing Protein-Protein Interactions.

    PubMed

    Zhou, Mi; Li, Qing; Wang, Renxiao

    2016-04-19

    Protein molecules often interact with other partner protein molecules in order to execute their vital functions in living organisms. Characterization of protein-protein interactions thus plays a central role in understanding the molecular mechanism of relevant protein molecules, elucidating the cellular processes and pathways relevant to health or disease for drug discovery, and charting large-scale interaction networks in systems biology research. A whole spectrum of methods, based on biophysical, biochemical, or genetic principles, have been developed to detect the time, space, and functional relevance of protein-protein interactions at various degrees of affinity and specificity. This article presents an overview of these experimental methods, outlining the principles, strengths and limitations, and recent developments of each type of method. PMID:26864455

  11. Protein – Which is Best?

    PubMed Central

    Hoffman, Jay R.; Falvo, Michael J.

    2004-01-01

    Protein intake that exceeds the recommended daily allowance is widely accepted for both endurance and power athletes. However, considering the variety of proteins that are available much less is known concerning the benefits of consuming one protein versus another. The purpose of this paper is to identify and analyze key factors in order to make responsible recommendations to both the general and athletic populations. Evaluation of a protein is fundamental in determining its appropriateness in the human diet. Proteins that are of inferior content and digestibility are important to recognize and restrict or limit in the diet. Similarly, such knowledge will provide an ability to identify proteins that provide the greatest benefit and should be consumed. The various techniques utilized to rate protein will be discussed. Traditionally, sources of dietary protein are seen as either being of animal or vegetable origin. Animal sources provide a complete source of protein (i.e. containing all essential amino acids), whereas vegetable sources generally lack one or more of the essential amino acids. Animal sources of dietary protein, despite providing a complete protein and numerous vitamins and minerals, have some health professionals concerned about the amount of saturated fat common in these foods compared to vegetable sources. The advent of processing techniques has shifted some of this attention and ignited the sports supplement marketplace with derivative products such as whey, casein and soy. Individually, these products vary in quality and applicability to certain populations. The benefits that these particular proteins possess are discussed. In addition, the impact that elevated protein consumption has on health and safety issues (i.e. bone health, renal function) are also reviewed. Key Points Higher protein needs are seen in athletic populations. Animal proteins is an important source of protein, however potential health concerns do exist from a diet of protein

  12. Protein-protein interactions of mitochondrial-associated protein via bioluminescence resonance energy transfer

    PubMed Central

    Koshiba, Takumi

    2015-01-01

    Protein-protein interactions are essential biological reactions occurring at inter- and intra-cellular levels. The analysis of their mechanism is generally required in order link to understand their various cellular functions. Bioluminescence resonance energy transfer (BRET), which is based on an enzymatic activity of luciferase, is a useful tool for investigating protein-protein interactions in live cells. The combination of the BRET system and biomolecular fluorescence complementation (BiFC) would provide us a better understanding of the hetero-oligomeric structural states of protein complexes. In this review, we discuss the application of BRET to the protein-protein interactions of mitochondrial-associated proteins and discuss its physiological relevance. PMID:27493852

  13. Membrane Bending by Protein Crowding

    NASA Astrophysics Data System (ADS)

    Stachowiak, Jeanne

    2014-03-01

    From endosomes and synaptic vesicles to the cristae of the mitochondria and the annulus of the nuclear pore, highly curved membranes are fundamental to the structure and physiology of living cells. The established view is that specific families of proteins are able to bend membranes by binding to them. For example, inherently curved proteins are thought to impose their structure on the membrane surface, while membrane-binding proteins with hydrophobic motifs are thought to insert into the membrane like wedges, driving curvature. However, computational models have recently revealed that these mechanisms would require specialized membrane-bending proteins to occupy nearly 100% of a curved membrane surface, an improbable physiological situation given the immense density and diversity of membrane-bound proteins, and the low expression levels of these specialized proteins within curved regions of the membrane. How then does curvature arise within the complex and crowded environment of cellular membranes? Our recent work using proteins involved in clathrin-mediated endocytosis, as well as engineered protein-lipid interactions, has suggested a new hypothesis - that lateral pressure generated by collisions between membrane-bound proteins can drive membrane bending. Specifically, by correlating membrane bending with quantitative optical measurements of protein density on synthetic membrane surfaces and simple physical models of collisions among membrane-bound proteins, we have demonstrated that protein-protein steric interactions can drive membrane curvature. These findings suggest that a simple imbalance in the concentration of membrane-bound proteins across a membrane surface can drive a membrane to bend, providing an efficient mechanism by which essentially any protein can contribute to shaping membranes.

  14. 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

  15. Green fluorescent protein: A perspective

    PubMed Central

    Remington, S James

    2011-01-01

    A brief personal perspective is provided for green fluorescent protein (GFP), covering the period 1994–2011. The topics discussed are primarily those in which my research group has made a contribution and include structure and function of the GFP polypeptide, the mechanism of fluorescence emission, excited state protein transfer, the design of ratiometric fluorescent protein biosensors and an overview of the fluorescent proteins derived from coral reef animals. Structure-function relationships in photoswitchable fluorescent proteins and nonfluorescent chromoproteins are also briefly covered. PMID:21714025

  16. Information contained in protein shapes

    NASA Technical Reports Server (NTRS)

    Sundaram, K.; Viswanadhan, V. N.; Macelroy, R. D.

    1983-01-01

    The sequence of local conformations at C-alpha atoms of a protein has been considered as an informational message string. The total self-information contents and self-information per letter have been evaluated for 83 globular proteins whose structures are known from X-ray crystallography. The derived information contents provide a method of quantitating structural specificity of proteins. This method of analysis enables repeating, intricate structural features to be recognized. Among the globular proteins whose structures have been solved, high potential iron protein stands out with the largest three-letter dependence.

  17. Selective chemical labeling of proteins.

    PubMed

    Chen, Xi; Wu, Yao-Wen

    2016-06-28

    Over the years, there have been remarkable efforts in the development of selective protein labeling strategies. In this review, we deliver a comprehensive overview of the currently available bioorthogonal and chemoselective reactions. The ability to introduce bioorthogonal handles to proteins is essential to carry out bioorthogonal reactions for protein labeling in living systems. We therefore summarize the techniques that allow for site-specific "installation" of bioorthogonal handles into proteins. We also highlight the biological applications that have been achieved by selective chemical labeling of proteins. PMID:26940577

  18. 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

  19. 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

  20. 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

  1. Intrinsic Localized Modes in Proteins.

    PubMed

    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

  2. New strategy to control cell migration and metastasis regulated by CCN2/CTGF

    PubMed Central

    2014-01-01

    Connective tissue growth factor (CTGF)/CCN family member 2 (CCN2) is a CCN family member of matricellular signaling modulators. It has been shown that CCN2/CTGF mediates cell adhesion, aggregation and migration in a large variety of cell types, including vascular endothelial cells, fibroblasts, epithelial cells, aortic smooth muscle and also pluripotent stem cells. Others matricellular proteins are capable of interacting with CCN2/CTGF to mediate its function. Cell migration is a key feature for tumor cell invasion and metastasis. CCN2/CTGF seems to be a prognostic marker for cancer. In addition, here we intend to discuss recent discoveries and a new strategy to develop therapies against CCN2/CTGF, in order to treat cancer metastasis. PMID:25120383

  3. 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.

  4. 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

  5. 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

  6. 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

  7. 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.

  8. 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.

  9. Protein Repeats from First Principles

    NASA Astrophysics Data System (ADS)

    Turjanski, Pablo; Parra, R. Gonzalo; Espada, Rocío; Becher, Verónica; Ferreiro, Diego U.

    2016-04-01

    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.

  10. Bioinformatic Prediction of WSSV-Host Protein-Protein Interaction

    PubMed Central

    Sun, Zheng; Xiang, Jianhai

    2014-01-01

    WSSV is one of the most dangerous pathogens in shrimp aquaculture. However, the molecular mechanism of how WSSV interacts with shrimp is still not very clear. In the present study, bioinformatic approaches were used to predict interactions between proteins from WSSV and shrimp. The genome data of WSSV (NC_003225.1) and the constructed transcriptome data of F. chinensis were used to screen potentially interacting proteins by searching in protein interaction databases, including STRING, Reactome, and DIP. Forty-four pairs of proteins were suggested to have interactions between WSSV and the shrimp. Gene ontology analysis revealed that 6 pairs of these interacting proteins were classified into “extracellular region” or “receptor complex” GO-terms. KEGG pathway analysis showed that they were involved in the “ECM-receptor interaction pathway.” In the 6 pairs of interacting proteins, an envelope protein called “collagen-like protein” (WSSV-CLP) encoded by an early virus gene “wsv001” in WSSV interacted with 6 deduced proteins from the shrimp, including three integrin alpha (ITGA), two integrin beta (ITGB), and one syndecan (SDC). Sequence analysis on WSSV-CLP, ITGA, ITGB, and SDC revealed that they possessed the sequence features for protein-protein interactions. This study might provide new insights into the interaction mechanisms between WSSV and shrimp. PMID:24982879

  11. 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. PMID:25343279

  12. LEA proteins prevent protein aggregation due to water stress

    PubMed Central

    Goyal, Kshamata; Walton, Laura J.; Tunnacliffe, Alan

    2005-01-01

    LEA (late embryogenesis abundant) proteins in both plants and animals are associated with tolerance to water stress resulting from desiccation and cold shock. However, although various functions of LEA proteins have been proposed, their precise role has not been defined. Recent bioinformatics studies suggest that LEA proteins might behave as molecular chaperones, and the current study was undertaken to test this hypothesis. Recombinant forms of AavLEA1, a group 3 LEA protein from the anhydrobiotic nematode Aphelenchus avenae, and Em, a group 1 LEA protein from wheat, have been subjected to functional analysis. Heat-stress experiments with citrate synthase, which is susceptible to aggregation at high temperatures, suggest that LEA proteins do not behave as classical molecular chaperones, but they do exhibit a protective, synergistic effect in the presence of the so-called chemical chaperone, trehalose. In contrast, both LEA proteins can independently protect citrate synthase from aggregation due to desiccation and freezing, in keeping with a role in water-stress tolerance; similar results were obtained with lactate dehydrogenase. This is the first evidence of anti-aggregation activity of LEA proteins due to water stress. Again, a synergistic effect of LEA and trehalose was observed, which is significant given that non-reducing disaccharides are known to accumulate during dehydration in plants and nematodes. A model is proposed whereby LEA proteins might act as a novel form of molecular chaperone, or ‘molecular shield’, to help prevent the formation of damaging protein aggregates during water stress. PMID:15631617

  13. 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

  14. 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.

  15. 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

  16. 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. PMID:24419610

  17. Chirality and protein folding

    NASA Astrophysics Data System (ADS)

    Kwiecinska, Joanna I.; Cieplak, Marek

    2005-05-01

    There are several simple criteria of folding to a native state in model proteins. One of them involves crossing of a threshold value of the root mean square deviation distance away from the native state. Another checks whether all native contacts are established, i.e. whether the interacting amino acids come closer than some characteristic distance. We use Go-like models of proteins and show that such simple criteria may prompt one to declare folding even though fragments of the resulting conformations have a wrong sense of chirality. We propose that a better condition of folding should augment the simple criteria with the requirement that most of the local values of the chirality should be nearly native. The kinetic discrepancy between the simple and compound criteria can be substantially reduced in the Go-like models by providing the Hamiltonian with a term which favours native values of the local chirality. We study the effects of this term as a function of its amplitude and compare it to other models such as ones with side groups and ones with angle-dependent potentials.

  18. Hyperquenching for protein cryocrystallography

    PubMed Central

    Warkentin, Matthew; Berejnov, Viatcheslav; Husseini, Naji S.; Thorne, Robert E.

    2010-01-01

    When samples having volumes characteristic of protein crystals are plunge cooled in liquid nitrogen or propane, most cooling occurs in the cold gas layer above the liquid. By removing this cold gas layer, cooling rates for small samples and modest plunge velocities are increased to 1.5 × 104 K s−1, with increases of a factor of 100 over current best practice possible with 10 μm samples. Glycerol concentrations required to eliminate water crystallization in protein-free aqueous mixtures drop from ∼28% w/v to as low as 6% w/v. These results will allow many crystals to go from crystallization tray to liquid cryogen to X-ray beam without cryoprotectants. By reducing or eliminating the need for cryoprotectants in growth solutions, they may also simplify the search for crystallization conditions and for optimal screens. The results presented here resolve many puzzles, such as why plunge cooling in liquid nitrogen or propane has, until now, not yielded significantly better diffraction quality than gas-stream cooling. PMID:20461232

  19. Water-transporting proteins.

    PubMed

    Zeuthen, Thomas

    2010-04-01

    Transport through lipids and aquaporins is osmotic and entirely driven by the difference in osmotic pressure. Water transport in cotransporters and uniporters is different: Water can be cotransported, energized by coupling to the substrate flux by a mechanism closely associated with protein. In the K(+)/Cl(-) and the Na(+)/K(+)/2Cl(-) cotransporters, water is entirely cotransported, while water transport in glucose uniporters and Na(+)-coupled transporters of nutrients and neurotransmitters takes place by both osmosis and cotransport. The molecular mechanism behind cotransport of water is not clear. It is associated with the substrate movements in aqueous pathways within the protein; a conventional unstirred layer mechanism can be ruled out, due to high rates of diffusion in the cytoplasm. The physiological roles of the various modes of water transport are reviewed in relation to epithelial transport. Epithelial water transport is energized by the movements of ions, but how the coupling takes place is uncertain. All epithelia can transport water uphill against an osmotic gradient, which is hard to explain by simple osmosis. Furthermore, genetic removal of aquaporins has not given support to osmosis as the exclusive mode of transport. Water cotransport can explain the coupling between ion and water transport, a major fraction of transepithelial water transport and uphill water transport. Aquaporins enhance water transport by utilizing osmotic gradients and cause the osmolarity of the transportate to approach isotonicity. PMID:20091162

  20. 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.

  1. Protein-protein interactions and genetic diseases: The Interactome

    PubMed Central

    Lage, Kasper

    2014-01-01

    Protein-protein interactions mediate essentially all biological processes. Despite the quality of these data being widely questioned a decade ago, the reproducibility of large-scale protein interaction data is now much improved and there is little question that the latest screens are of high quality. Moreover, common data standards and coordinated curation practices between the databases that collect the interactions have made these valuable data available to a wide group of researchers. Here, I will review how protein-protein interactions are measured, collected and quality controlled. I discuss how the architecture of molecular protein networks have informed disease biology, and how these data are now being computationally integrated with the newest genomic technologies, in particular genome-wide association studies and exome-sequencing projects, to improve our understanding of molecular processes perturbed by genetics in human diseases. PMID:24892209

  2. 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

  3. Mitochondrial nucleoid interacting proteins support mitochondrial protein synthesis

    PubMed Central

    He, J.; Cooper, H. M.; Reyes, A.; Di Re, M.; Sembongi, H.; Litwin, T. R.; Gao, J.; Neuman, K. C.; Fearnley, I. M.; Spinazzola, A.; Walker, J. E.; Holt, I. J.

    2012-01-01

    Mitochondrial ribosomes and translation factors co-purify with mitochondrial nucleoids of human cells, based on affinity protein purification of tagged mitochondrial DNA binding proteins. Among the most frequently identified proteins were ATAD3 and prohibitin, which have been identified previously as nucleoid components, using a variety of methods. Both proteins are demonstrated to be required for mitochondrial protein synthesis in human cultured cells, and the major binding partner of ATAD3 is the mitochondrial ribosome. Altered ATAD3 expression also perturbs mtDNA maintenance and replication. These findings suggest an intimate association between nucleoids and the machinery of protein synthesis in mitochondria. ATAD3 and prohibitin are tightly associated with the mitochondrial membranes and so we propose that they support nucleic acid complexes at the inner membrane of the mitochondrion. PMID:22453275

  4. 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.

  5. Revisiting the Voronoi description of protein-protein interfaces.

    PubMed

    Cazals, Frédéric; Proust, Flavien; Bahadur, Ranjit P; Janin, Joël

    2006-09-01

    We developed a model of macromolecular interfaces based on the Voronoi diagram and the related alpha-complex, and we tested its properties on a set of 96 protein-protein complexes taken from the Protein Data Bank. The Voronoi model provides a natural definition of the interfaces, and it yields values of the number of interface atoms and of the interface area that have excellent correlation coefficients with those of the classical model based on solvent accessibility. Nevertheless, some atoms that do not lose solvent accessibility are part of the interface defined by the Voronoi model. The Voronoi model provides robust definitions of the curvature and of the connectivity of the interfaces, and leads to estimates of these features that generally agree with other approaches. Our implementation of the model allows an analysis of protein-water contacts that highlights the role of structural water molecules at protein-protein interfaces. PMID:16943442

  6. Signature Product Code for Predicting Protein-Protein Interactions

    Energy Science and Technology Software Center (ESTSC)

    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

  7. Proteins interacting with cloning scars: a source of false positive protein-protein interactions

    PubMed Central

    Banks, Charles A. S.; Boanca, Gina; Lee, Zachary T.; Florens, Laurence; Washburn, Michael P.

    2015-01-01

    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. PMID:25704442

  8. Characterization of Protein Complexes and Subcomplexes in Protein-Protein Interaction Databases

    PubMed Central

    Zaki, Nazar; Mohamed, Elfadil A.; Mora, Antonio

    2015-01-01

    The identification and characterization of protein complexes implicated in protein-protein interaction data are crucial to the understanding of the molecular events under normal and abnormal physiological conditions. This paper provides a novel characterization of subcomplexes in protein interaction databases, stressing definition and representation issues, quantification, biological validation, network metrics, motifs, modularity, and gene ontology (GO) terms. The paper introduces the concept of “nested group” as a way to represent subcomplexes and estimates that around 15% of those nested group with the higher Jaccard index may be a result of data artifacts in protein interaction databases, while a number of them can be found in biologically important modular structures or dynamic structures. We also found that network centralities, enrichment in essential proteins, GO terms related to regulation, imperfect 5-clique motifs, and higher GO homogeneity can be used to identify proteins in nested complexes. PMID:25722891

  9. A computational system for modelling flexible protein-protein and protein-DNA docking.

    PubMed

    Sternberg, M J; Aloy, P; Gabb, H A; Jackson, R M; Moont, G; Querol, E; Aviles, F X

    1998-01-01

    A computational system is described that predicts the structure of protein/protein and protein/DNA complexes starting from unbound coordinate sets. The approach is (i) a global search with rigid-body docking for complexes with shape complementarity and favourable electrostatics; (ii) use of distance constraints from experimental (or predicted) knowledge of critical residues; (iii) use of pair potential to screen docked complexes and (iv) refinement and further screening by protein-side chain optimisation and interfacial energy minimisation. The system has been applied to model ten protein/protein and eight protein-repressor/DNA (steps i to iii only) complexes. In general a few complexes, one of which is close to the true structure, can be generated. PMID:9783224

  10. Proteins aggregation and human diseases

    NASA Astrophysics Data System (ADS)

    Hu, Chin-Kun

    2015-04-01

    Many human diseases and the death of most supercentenarians are related to protein aggregation. Neurodegenerative diseases include Alzheimer's disease (AD), Huntington's disease (HD), Parkinson's disease (PD), frontotemporallobar degeneration, etc. Such diseases are due to progressive loss of structure or function of neurons caused by protein aggregation. For example, AD is considered to be related to aggregation of Aβ40 (peptide with 40 amino acids) and Aβ42 (peptide with 42 amino acids) and HD is considered to be related to aggregation of polyQ (polyglutamine) peptides. In this paper, we briefly review our recent discovery of key factors for protein aggregation. We used a lattice model to study the aggregation rates of proteins and found that the probability for a protein sequence to appear in the conformation of the aggregated state can be used to determine the temperature at which proteins can aggregate most quickly. We used molecular dynamics and simple models of polymer chains to study relaxation and aggregation of proteins under various conditions and found that when the bending-angle dependent and torsion-angle dependent interactions are zero or very small, then protein chains tend to aggregate at lower temperatures. All atom models were used to identify a key peptide chain for the aggregation of insulin chains and to find that two polyQ chains prefer anti-parallel conformation. It is pointed out that in many cases, protein aggregation does not result from protein mis-folding. A potential drug from Chinese medicine was found for Alzheimer's disease.

  11. Protein Adaptations in Archaeal Extremophiles

    PubMed Central

    Reed, Christopher J.; Lewis, Hunter; Trejo, Eric; Winston, Vern; Evilia, Caryn

    2013-01-01

    Extremophiles, especially those in Archaea, have a myriad of adaptations that keep their cellular proteins stable and active under the extreme conditions in which they live. Rather than having one basic set of adaptations that works for all environments, Archaea have evolved separate protein features that are customized for each environment. We categorized the Archaea into three general groups to describe what is known about their protein adaptations: thermophilic, psychrophilic, and halophilic. Thermophilic proteins tend to have a prominent hydrophobic core and increased electrostatic interactions to maintain activity at high temperatures. Psychrophilic proteins have a reduced hydrophobic core and a less charged protein surface to maintain flexibility and activity under cold temperatures. Halophilic proteins are characterized by increased negative surface charge due to increased acidic amino acid content and peptide insertions, which compensates for the extreme ionic conditions. While acidophiles, alkaliphiles, and piezophiles are their own class of Archaea, their protein adaptations toward pH and pressure are less discernible. By understanding the protein adaptations used by archaeal extremophiles, we hope to be able to engineer and utilize proteins for industrial, environmental, and biotechnological applications where function in extreme conditions is required for activity. PMID:24151449

  12. Laboratory-Directed Protein Evolution

    PubMed Central

    Yuan, Ling; Kurek, Itzhak; English, James; Keenan, Robert

    2005-01-01

    Systematic approaches to directed evolution of proteins have been documented since the 1970s. The ability to recruit new protein functions arises from the considerable substrate ambiguity of many proteins. The substrate ambiguity of a protein can be interpreted as the evolutionary potential that allows a protein to acquire new specificities through mutation or to regain function via mutations that differ from the original protein sequence. All organisms have evolutionarily exploited this substrate ambiguity. When exploited in a laboratory under controlled mutagenesis and selection, it enables a protein to “evolve” in desired directions. One of the most effective strategies in directed protein evolution is to gradually accumulate mutations, either sequentially or by recombination, while applying selective pressure. This is typically achieved by the generation of libraries of mutants followed by efficient screening of these libraries for targeted functions and subsequent repetition of the process using improved mutants from the previous screening. Here we review some of the successful strategies in creating protein diversity and the more recent progress in directed protein evolution in a wide range of scientific disciplines and its impacts in chemical, pharmaceutical, and agricultural sciences. PMID:16148303

  13. EH domain proteins regulate cardiac membrane protein targeting

    PubMed Central

    Gudmundsson, Hjalti; Hund, Thomas J.; Wright, Patrick J.; Kline, Crystal F.; Snyder, Jedidiah S.; Qian, Lan; Koval, Olha M.; Cunha, Shane R.; George, Manju; Rainey, Mark A.; Kashef, Farshid E.; Dun, Wen; Boyden, Penelope A.; Anderson, Mark E.; Band, Hamid; Mohler, Peter J.

    2010-01-01

    Rationale Cardiac membrane excitability is tightly regulated by an integrated network of membrane-associated ion channels, transporters, receptors, and signaling molecules. Membrane protein dynamics in health and disease are maintained by a complex ensemble of intracellular targeting, scaffolding, recycling, and degradation pathways. Surprisingly, despite decades of research linking dysfunction in membrane protein trafficking with human cardiovascular disease, essentially nothing is known regarding the molecular identity or function of these intracellular targeting pathways in excitable cardiomyocytes. Objective We sought to discover novel pathways for membrane protein targeting in primary cardiomyocytes. Methods and Results We report the initial characterization of a large family of membrane trafficking proteins in human heart. We employed a tissue-wide screen for novel ankyrin-associated trafficking proteins and identified four members of a unique Eps15 homology (EH) domain-containing protein family (EHD1, EHD2, EHD3, EHD4) that serve critical roles in endosome-based membrane protein targeting in other cell types. We show that EHD1-4 directly associate with ankyrin, provide the first information on the expression and localization of these molecules in primary cardiomyocytes, and demonstrate that EHD1-4 are co-expressed with ankyrin-B in the myocyte perinuclear region. Notably, the expression of multiple EHD proteins is increased in animal models lacking ankyrin-B, and EHD3-deficient cardiomyocytes display aberrant ankyrin-B localization and selective loss of Na/Ca exchanger expression and function. Finally, we report significant modulation of EHD expression following myocardial infarction, suggesting that these proteins may play a key role in regulating membrane excitability in normal and diseased heart. Conclusions Our findings identify and characterize a new class of cardiac trafficking proteins, define the first group of proteins associated with the ankyrin

  14. 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

  15. Essential protein identification based on essential protein-protein interaction prediction by Integrated Edge Weights.

    PubMed

    Jiang, Yuexu; Wang, Yan; Pang, Wei; Chen, Liang; Sun, Huiyan; Liang, Yanchun; Blanzieri, Enrico

    2015-07-15

    Essential proteins play a crucial role in cellular survival and development process. Experimentally, essential proteins are identified by gene knockouts or RNA interference, which are expensive and often fatal to the target organisms. Regarding this, an alternative yet important approach to essential protein identification is through computational prediction. Existing computational methods predict essential proteins based on their relative densities in a protein-protein interaction (PPI) network. Degree, betweenness, and other appropriate criteria are often used to measure the relative density. However, no matter what criterion is used, a protein is actually ordered by the attributes of this protein per se. In this research, we presented a novel computational method, Integrated Edge Weights (IEW), to first rank protein-protein interactions by integrating their edge weights, and then identified sub PPI networks consisting of those highly-ranked edges, and finally regarded the nodes in these sub networks as essential proteins. We evaluated IEW on three model organisms: Saccharomyces cerevisiae (S. cerevisiae), Escherichia coli (E. coli), and Caenorhabditis elegans (C. elegans). The experimental results showed that IEW achieved better performance than the state-of-the-art methods in terms of precision-recall and Jackknife measures. We had also demonstrated that IEW is a robust and effective method, which can retrieve biologically significant modules by its highly-ranked protein-protein interactions for S. cerevisiae, E. coli, and C. elegans. We believe that, with sufficient data provided, IEW can be used to any other organisms' essential protein identification. A website about IEW can be accessed from http://digbio.missouri.edu/IEW/index.html. PMID:25892709

  16. Protein degradation and protection against misfolded or damaged proteins

    NASA Astrophysics Data System (ADS)

    Goldberg, Alfred L.

    2003-12-01

    The ultimate mechanism that cells use to ensure the quality of intracellular proteins is the selective destruction of misfolded or damaged polypeptides. In eukaryotic cells, the large ATP-dependent proteolytic machine, the 26S proteasome, prevents the accumulation of non-functional, potentially toxic proteins. This process is of particular importance in protecting cells against harsh conditions (for example, heat shock or oxidative stress) and in a variety of diseases (for example, cystic fibrosis and the major neurodegenerative diseases). A full understanding of the pathogenesis of the protein-folding diseases will require greater knowledge of how misfolded proteins are recognized and selectively degraded.

  17. Membrane Protein Solubilization and Composition of Protein Detergent Complexes.

    PubMed

    Duquesne, Katia; Prima, Valérie; Sturgis, James N

    2016-01-01

    Membrane proteins are typically expressed in heterologous systems with a view to in vitro characterization. A critical step in the preparation of membrane proteins after expression in any system is the solubilization of the protein in aqueous solution, typically using detergents and lipids, to obtain the protein in a form suitable for purification, structural or functional analysis. This process is particularly difficult as the objective is to prepare the protein in an unnatural environment, a protein detergent complex, separating it from its natural lipid partners while causing the minimum destabilization or modification of the structure. Although the process is difficult, and relatively hard to master, an increasing number of membrane proteins have been successfully isolated after expression in a wide variety of systems. In this chapter we give a general protocol for preparing protein detergent complexes that is aimed at guiding the reader through the different critical steps. In the second part of the chapter we illustrate how to analyze the composition of protein detergent complexes; this analysis is important as it has been found that compositional variation often causes irreproducible results. PMID:27485340

  18. Protein-protein interactions and prediction: a comprehensive overview.

    PubMed

    Sowmya, Gopichandran; Ranganathan, Shoba

    2014-01-01

    Molecular function in cellular processes is governed by protein-protein interactions (PPIs) within biological networks. Selective yet specific association of these protein partners contributes to diverse functionality such as catalysis, regulation, assembly, immunity, and inhibition in a cell. Therefore, understanding the principles of protein-protein association has been of immense interest for several decades. We provide an overview of the experimental methods used to determine PPIs and the key databases archiving this information. Structural and functional information of existing protein complexes confers knowledge on the principles of PPI, based on which a classification scheme for PPIs is then introduced. Obtaining high-quality non-redundant datasets of protein complexes for interaction characterisation is an essential step towards deciphering their underlying binding principles. Analysis of physicochemical features and their documentation has enhanced our understanding of the molecular basis of protein-protein association. We describe the diverse datasets created/collected by various groups and their key findings inferring distinguishing features. The currently available interface databases and prediction servers have also been compiled. PMID:23855658

  19. Novel computational methods to design protein-protein interactions

    NASA Astrophysics Data System (ADS)

    Zhou, Alice Qinhua; O'Hern, Corey; Regan, Lynne

    2014-03-01

    Despite the abundance of structural data, we still cannot accurately predict the structural and energetic changes resulting from mutations at protein interfaces. The inadequacy of current computational approaches to the analysis and design of protein-protein interactions has hampered the development of novel therapeutic and diagnostic agents. In this work, we apply a simple physical model that includes only a minimal set of geometrical constraints, excluded volume, and attractive van der Waals interactions to 1) rank the binding affinity of mutants of tetratricopeptide repeat proteins with their cognate peptides, 2) rank the energetics of binding of small designed proteins to the hydrophobic stem region of the influenza hemagglutinin protein, and 3) predict the stability of T4 lysozyme and staphylococcal nuclease mutants. This work will not only lead to a fundamental understanding of protein-protein interactions, but also to the development of efficient computational methods to rationally design protein interfaces with tunable specificity and affinity, and numerous applications in biomedicine. NSF DMR-1006537, PHY-1019147, Raymond and Beverly Sackler Institute for Biological, Physical and Engineering Sciences, and Howard Hughes Medical Institute.

  20. Nanosecond Relaxation Dynamics of Hydrated Proteins: Water versus protein contributions

    SciTech Connect

    Khodadadi, S; Curtis, J. E.; Sokolov, Alexei P

    2011-01-01

    We have studied picosecond to nanosecond dynamics of hydrated protein powders using dielectric spectroscopy and molecular dynamics (MD) simulations. Our analysis of hydrogen-atom single particle dynamics from MD simulations focused on main ( main tens of picoseconds) and slow ( slow nanosecond) relaxation processes that were observed in dielectric spectra of similar hydrated protein samples. Traditionally, the interpretation of these processes observed in dielectric spectra has been ascribed to the relaxation behavior of hydration water tightly bounded to a protein and not to protein atoms. Detailed analysis of the MD simulations and comparison to dielectric data indicate that the observed relaxation process in the nanosecond time range of hydrated protein spectra is mainly due to protein atoms. The relaxation processes involve the entire structure of protein including atoms in the protein backbone, side chains, and turns. Both surface and buried protein atoms contribute to the slow processes; however, surface atoms demonstrate slightly faster relaxation dynamics. Analysis of the water molecule residence and dipolar relaxation correlation behavior indicates that the hydration water relaxes at much shorter time scales.

  1. Protein-protein interface prediction based on hexagon structure similarity.

    PubMed

    Guo, Fei; Ding, Yijie; Li, Shuai Cheng; Shen, Chao; Wang, Lusheng

    2016-08-01

    Studies on protein-protein interaction are important in proteome research. How to build more effective models based on sequence information, structure information and physicochemical characteristics, is the key technology in protein-protein interface prediction. In this paper, we study the protein-protein interface prediction problem. We propose a novel method for identifying residues on interfaces from an input protein with both sequence and 3D structure information, based on hexagon structure similarity. Experiments show that our method achieves better results than some state-of-the-art methods for identifying protein-protein interface. Comparing to existing methods, our approach improves F-measure value by at least 0.03. On a common dataset consisting of 41 complexes, our method has overall precision and recall values of 63% and 57%. On Benchmark v4.0, our method has overall precision and recall values of 55% and 56%. On CAPRI targets, our method has overall precision and recall values of 52% and 55%. PMID:26936323

  2. Noninvasive imaging of protein-protein interactions in living animals

    NASA Astrophysics Data System (ADS)

    Luker, Gary D.; Sharma, Vijay; Pica, Christina M.; Dahlheimer, Julie L.; Li, Wei; Ochesky, Joseph; Ryan, Christine E.; Piwnica-Worms, Helen; Piwnica-Worms, David

    2002-05-01

    Protein-protein interactions control transcription, cell division, and cell proliferation as well as mediate signal transduction, oncogenic transformation, and regulation of cell death. Although a variety of methods have been used to investigate protein interactions in vitro and in cultured cells, none can analyze these interactions in intact, living animals. To enable noninvasive molecular imaging of protein-protein interactions in vivo by positron-emission tomography and fluorescence imaging, we engineered a fusion reporter gene comprising a mutant herpes simplex virus 1 thymidine kinase and green fluorescent protein for readout of a tetracycline-inducible, two-hybrid system in vivo. By using micro-positron-emission tomography, interactions between p53 tumor suppressor and the large T antigen of simian virus 40 were visualized in tumor xenografts of HeLa cells stably transfected with the imaging constructs. Imaging protein-binding partners in vivo will enable functional proteomics in whole animals and provide a tool for screening compounds targeted to specific protein-protein interactions in living animals.

  3. Methods for analyzing and quantifying protein-protein interaction.

    PubMed

    Syafrizayanti; Betzen, Christian; Hoheisel, Jörg D; Kastelic, Damjana

    2014-02-01

    Genome sequencing has led to the identification of many proteins, which had not been recognized before. In consequence, the basic set of human proteins is generally known. Far less information, however, exists about protein-protein interactions, which are required and responsible for cellular activities and their control. Many protein isoforms that result from mutations, splice-variations and post-translational modifications also come into play. Until recently, interactions of only few protein partners could be analyzed in a single experiment. However, this does not meet the challenge of investigating the highly complex interaction patterns in cellular systems. It is made even more demanding by the need to determine the intensity of interactions quantitatively in order to properly understand protein interplay. Currently available techniques vary with respect to accuracy, reliability, reproducibility and throughput and their performances range from a mere qualitative demonstration of binding to a quantitative characterization of affinities. In this article, an overview is given of the methodologies available for analysis of protein-protein interactions. PMID:24393018

  4. Cry Protein Crystals: A Novel Platform for Protein Delivery

    PubMed Central

    Bonnegarde-Bernard, Astrid; Wallace, Julie A.; Dean, Donald H.; Ostrowski, Michael C.; Burry, Richard W.; Boyaka, Prosper N.; Chan, Michael K.

    2015-01-01

    Protein delivery platforms are important tools in the development of novel protein therapeutics and biotechnologies. We have developed a new class of protein delivery agent based on sub-micrometer-sized Cry3Aa protein crystals that naturally form within the bacterium Bacillus thuringiensis. We demonstrate that fusion of the cry3Aa gene to that of various reporter proteins allows for the facile production of Cry3Aa fusion protein crystals for use in subsequent applications. These Cry3Aa fusion protein crystals are efficiently taken up and retained by macrophages and other cell lines in vitro, and can be delivered to mice in vivo via multiple modes of administration. Oral delivery of Cry3Aa fusion protein crystals to C57BL/6 mice leads to their uptake by MHC class II cells, including macrophages in the Peyer’s patches, supporting the notion that the Cry3Aa framework can be used to stabilize cargo protein against degradation for delivery to gastrointestinal lymphoid tissues. PMID:26030844

  5. Protein Crystal Growth

    NASA Technical Reports Server (NTRS)

    2003-01-01

    In order to rapidly and efficiently grow crystals, tools were needed to automatically identify and analyze the growing process of protein crystals. To meet this need, Diversified Scientific, Inc. (DSI), with the support of a Small Business Innovation Research (SBIR) contract from NASA s Marshall Space Flight Center, developed CrystalScore(trademark), the first automated image acquisition, analysis, and archiving system designed specifically for the macromolecular crystal growing community. It offers automated hardware control, image and data archiving, image processing, a searchable database, and surface plotting of experimental data. CrystalScore is currently being used by numerous pharmaceutical companies and academic and nonprofit research centers. DSI, located in Birmingham, Alabama, was awarded the patent Method for acquiring, storing, and analyzing crystal images on March 4, 2003. Another DSI product made possible by Marshall SBIR funding is VaporPro(trademark), a unique, comprehensive system that allows for the automated control of vapor diffusion for crystallization experiments.

  6. Carotenoid-Protein Interactions

    NASA Astrophysics Data System (ADS)

    Britton, George; Helliwell, John R.

    Chapter 5 shows that the aggregation of carotenoid molecules can have a profound effect on their properties and hence their functioning in biological systems. Another important influence is the interaction between carotenoids and other molecules. The way that interactions of carotenoids with lipid bilayers influence the structure and properties of membranes and membrane-asociated processes is discussed in Chapter 10, and the aggregation of carotenoid molecules within the bilayers in Chapter 5. Of particular importance, though, are interactions between carotenoids and proteins. These allow the hydrophobic carotenoids to be transported, to exist, and to function in an aqueous environment. In some cases they may modify strongly the light-absorption properties and hence the colour and photochemistry of the carotenoids.

  7. Protein detection system

    DOEpatents

    Fruetel, Julie A.; Fiechtner, Gregory J.; Kliner, Dahv A. V.; McIlroy, Andrew

    2009-05-05

    The present embodiment describes a miniature, microfluidic, absorption-based sensor to detect proteins at sensitivities comparable to LIF but without the need for tagging. This instrument utilizes fiber-based evanescent-field cavity-ringdown spectroscopy, in combination with faceted prism microchannels. The combination of these techniques will increase the effective absorption path length by a factor of 10.sup.3 to 10.sup.4 (to .about.1-m), thereby providing unprecedented sensitivity using direct absorption. The coupling of high-sensitivity absorption with high-performance microfluidic separation will enable real-time sensing of biological agents in aqueous samples (including aerosol collector fluids) and will provide a general method with spectral fingerprint capability for detecting specific bio-agents.

  8. Protein Hormones and Immunity‡

    PubMed Central

    Kelley, Keith W.; Weigent, Douglas A.; Kooijman, Ron

    2007-01-01

    A number of observations and discoveries over the past 20 years support the concept of important physiological interactions between the endocrine and immune systems. The best known pathway for transmission of information from the immune system to the neuroendocrine system is humoral in the form of cytokines, although neural transmission via the afferent vagus is well documented also. In the other direction, efferent signals from the nervous system to the immune system are conveyed by both the neuroendocrine and autonomic nervous systems. Communication is possible because the nervous and immune systems share a common biochemical language involving shared ligands and receptors, including neurotransmitters, neuropeptides, growth factors, neuroendocrine hormones and cytokines. This means that the brain functions as an immune-regulating organ participating in immune responses. A great deal of evidence has accumulated and confirmed that hormones secreted by the neuroendocrine system play an important role in communication and regulation of the cells of the immune system. Among protein hormones, this has been most clearly documented for prolactin (PRL), growth hormone (GH), and insulin-like growth factor-1 (IGF-I), but significant influences on immunity by thyroid stimulating hormone (TSH) have also been demonstrated. Here we review evidence obtained during the past 20 years to clearly demonstrate that neuroendocrine protein hormones influence immunity and that immune processes affect the neuroendocrine system. New findings highlight a previously undiscovered route of communication between the immune and endocrine systems that is now known to occur at the cellular level. This communication system is activated when inflammatory processes induced by proinflammatory cytokines antagonize the function of a variety of hormones, which then causes endocrine resistance in both the periphery and brain. Homeostasis during inflammation is achieved by a balance between cytokines and

  9. Protein extraction from activated sludge.

    PubMed

    Denecke, M

    2006-01-01

    Two methods for the separation of protein originating from activated sludge were compared. In one method, the total protein was isolated out of the activated sludge (crude extract). These samples included all dissolved proteins originating from the bacterial cells and biofilm made up of extracellular polymeric substances (EPS). Every time polyacrylamide gel electrophoresis (PAGE) was done, the protein bands from samples of crude extract were covered by polymeric substances including carbohydrates, uronic acids or humic compounds. Using the immunoblot technique it was possible to demonstrate the presence of the heat shock protein HSP70 in crude extracts of activated sludge. The comparison of protein fingerprints required that clear and distinct bands appear on the PAGE analysis. To this end, a procedure to separates bacterial cells from the EPS was developed. Bacterial cells were separated by incubation with EDTA and subsequent filtration. The isolated cells were directly incubated in a sample buffer. PMID:16898150

  10. Advantages of proteins being disordered

    PubMed Central

    Liu, Zhirong; Huang, Yongqi

    2014-01-01

    The past decade has witnessed great advances in our understanding of protein structure-function relationships in terms of the ubiquitous existence of intrinsically disordered proteins (IDPs) and intrinsically disordered regions (IDRs). The structural disorder of IDPs/IDRs enables them to play essential functions that are complementary to those of ordered proteins. In addition, IDPs/IDRs are persistent in evolution. Therefore, they are expected to possess some advantages over ordered proteins. In this review, we summarize and survey nine possible advantages of IDPs/IDRs: economizing genome/protein resources, overcoming steric restrictions in binding, achieving high specificity with low affinity, increasing binding rate, facilitating posttranslational modifications, enabling flexible linkers, preventing aggregation, providing resistance to non-native conditions, and allowing compatibility with more available sequences. Some potential advantages of IDPs/IDRs are not well understood and require both experimental and theoretical approaches to decipher. The connection with protein design is also briefly discussed. PMID:24532081

  11. Synthetic Peptides as Protein Mimics

    PubMed Central

    Groß, Andrea; Hashimoto, Chie; Sticht, Heinrich; Eichler, Jutta

    2016-01-01

    The design and generation of molecules capable of mimicking the binding and/or functional sites of proteins represents a promising strategy for the exploration and modulation of protein function through controlled interference with the underlying molecular interactions. Synthetic peptides have proven an excellent type of molecule for the mimicry of protein sites because such peptides can be generated as exact copies of protein fragments, as well as in diverse chemical modifications, which includes the incorporation of a large range of non-proteinogenic amino acids as well as the modification of the peptide backbone. Apart from extending the chemical and structural diversity presented by peptides, such modifications also increase the proteolytic stability of the molecules, enhancing their utility for biological applications. This article reviews recent advances by this and other laboratories in the use of synthetic protein mimics to modulate protein function, as well as to provide building blocks for synthetic biology. PMID:26835447

  12. Protein Degradation and Iron Homeostasis

    PubMed Central

    Thompson, Joel W.; Bruick, Richard K.

    2013-01-01

    Regulation of both systemic and cellular iron homeostasis requires the capacity to sense iron levels and appropriately modify the expression of iron metabolism genes. These responses are coordinated through the efforts of several key regulatory factors including F-box and Leucine-rich Repeat Protein 5 (FBXL5), Iron Regulatory Proteins (IRPs), Hypoxia Inducible Factor (HIF), and ferroportin. Notably, the stability of each of these proteins is regulated in response to iron. Recent discoveries have greatly advanced our understanding of the molecular mechanisms governing iron-sensing and protein degradation within these pathways. It has become clear that iron’s privileged roles in both enzyme catalysis and protein structure contribute to its regulation of protein stability. Moreover, these multiple pathways intersect with one another in larger regulatory networks to maintain iron homeostasis. PMID:22349011

  13. Protein degradation and iron homeostasis.

    PubMed

    Thompson, Joel W; Bruick, Richard K

    2012-09-01

    Regulation of both systemic and cellular iron homeostasis requires the capacity to sense iron levels and appropriately modify the expression of iron metabolism genes. These responses are coordinated through the efforts of several key regulatory factors including F-box and Leucine-rich Repeat Protein 5 (FBXL5), Iron Regulatory Proteins (IRPs), Hypoxia Inducible Factor (HIF), and ferroportin. Notably, the stability of each of these proteins is regulated in response to iron. Recent discoveries have greatly advanced our understanding of the molecular mechanisms governing iron-sensing and protein degradation within these pathways. It has become clear that iron's privileged roles in both enzyme catalysis and protein structure contribute to its regulation of protein stability. Moreover, these multiple pathways intersect with one another in larger regulatory networks to maintain iron homeostasis. This article is part of a Special Issue entitled: Cell Biology of Metals. PMID:22349011

  14. Quantum dots and prion proteins

    PubMed Central

    Sobrova, Pavlina; Blazkova, Iva; Chomoucka, Jana; Drbohlavova, Jana; Vaculovicova, Marketa; Kopel, Pavel; Hubalek, Jaromir; Kizek, Rene; Adam, Vojtech

    2013-01-01

    A diagnostics of infectious diseases can be done by the immunologic methods or by the amplification of nucleic acid specific to contagious agent using polymerase chain reaction. However, in transmissible spongiform encephalopathies, the infectious agent, prion protein (PrPSc), has the same sequence of nucleic acids as a naturally occurring protein. The other issue with the diagnosing based on the PrPSc detection is that the pathological form of prion protein is abundant only at late stages of the disease in a brain. Therefore, the diagnostics of prion protein caused diseases represent a sort of challenges as that hosts can incubate infectious prion proteins for many months or even years. Therefore, new in vivo assays for detection of prion proteins and for diagnosis of their relation to neurodegenerative diseases are summarized. Their applicability and future prospects in this field are discussed with particular aim at using quantum dots as fluorescent labels. PMID:24055838

  15. Biofoams and natural protein surfactants

    PubMed Central

    Cooper, Alan; Kennedy, Malcolm W.

    2010-01-01

    Naturally occurring foam constituent and surfactant proteins with intriguing structures and functions are now being identified from a variety of biological sources. The ranaspumins from tropical frog foam nests comprise a range of proteins with a mixture of surfactant, carbohydrate binding and antimicrobial activities that together provide a stable, biocompatible, protective foam environment for developing eggs and embryos. Ranasmurfin, a blue protein from a different species of frog, displays a novel structure with a unique chromophoric crosslink. Latherin, primarily from horse sweat, but with similarities to salivary, oral and upper respiratory tract proteins, illustrates several potential roles for surfactant proteins in mammalian systems. These proteins, together with the previously discovered hydrophobins of fungi, throw new light on biomolecular processes at air–water and other interfaces. This review provides a perspective on these recent findings, focussing on structure and biophysical properties. PMID:20615601

  16. The Cost of Protein Production

    PubMed Central

    Kafri, Moshe; Metzl-Raz, Eyal; Jona, Ghil; Barkai, Naama

    2015-01-01

    Summary The economy of protein production is central to cell physiology, being intimately linked with cell division rate and cell size. Attempts to model cellular physiology are limited by the scarcity of experimental data defining the molecular processes limiting protein expression. Here, we distinguish the relative contribution of gene transcription and protein translation to the slower proliferation of budding yeast producing excess levels of unneeded proteins. In contrast to widely held assumptions, rapidly growing cells are not universally limited by ribosome content. Rather, transcription dominates cost under some conditions (e.g., low phosphate), translation in others (e.g., low nitrogen), and both in other conditions (e.g., rich media). Furthermore, cells adapted to enforced protein production by becoming larger and increasing their endogenous protein levels, suggesting limited competition for common resources. We propose that rapidly growing cells do not exhaust their resources to maximize growth but maintain sufficient reserves to accommodate changing requirements. PMID:26725116

  17. Algorithmic complexity of a protein

    NASA Astrophysics Data System (ADS)

    Dewey, T. Gregory

    1996-07-01

    The information contained in a protein's amino acid sequence dictates its three-dimensional structure. To quantitate the transfer of information that occurs in the protein folding process, the Kolmogorov information entropy or algorithmic complexity of the protein structure is investigated. The algorithmic complexity of an object provides a means of quantitating its information content. Recent results have indicated that the algorithmic complexity of microstates of certain statistical mechanical systems can be estimated from the thermodynamic entropy. In the present work, it is shown that the algorithmic complexity of a protein is given by its configurational entropy. Using this result, a quantitative estimate of the information content of a protein's structure is made and is compared to the information content of the sequence. Additionally, the mutual information between sequence and structure is determined. It is seen that virtually all the information contained in the protein structure is shared with the sequence.

  18. Theoretical studies of protein-protein and protein-DNA binding rates

    NASA Astrophysics Data System (ADS)

    Alsallaq, Ramzi A.

    Proteins are folded chains of amino acids. Some of the amino acids (e.g. Lys, Arg, His, Asp, and Glu) carry charges under physiological conditions. Proteins almost always function through binding to other proteins or ligands, for example barnase is a ribonuclease protein, found in the bacterium Bacillus amyloliquefaceus. Barnase degrades RNA by hydrolysis. For the bacterium to inhibit the potentially lethal action of Barnase within its own cell it co-produces another protein called barstar which binds quickly, and tightly, to barnase. The biological function of this binding is to block the active site of barnase. The speeds (rates) at which proteins associate are vital to many biological processes. They span a wide range (from less than 103 to 108 M-1s-1 ). Rates greater than ˜ 106 M -1s-1 are typically found to be manifestations of enhancements by long-range electrostatic interactions between the associating proteins. A different paradigm appears in the case of protein binding to DNA. The rate in this case is enhanced through attractive surface potential that effectively reduces the dimensionality of the available search space for the diffusing protein. This thesis presents computational and theoretical models on the rate of association of ligands/proteins to other proteins or DNA. For protein-protein association we present a general strategy for computing protein-protein rates of association. The main achievements of this strategy is the ability to obtain a stringent reaction criteria based on the landscape of short-range interactions between the associating proteins, and the ability to compute the effect of the electrostatic interactions on the rates of association accurately using the best known solvers for Poisson-Boltzmann equation presently available. For protein-DNA association we present a mathematical model for proteins targeting specific sites on a circular DNA topology. The main achievements are the realization that a linear DNA with reflecting ends

  19. TGF-beta signaling proteins and the Protein Ontology

    PubMed Central

    Arighi, Cecilia N; Liu, Hongfang; Natale, Darren A; Barker, Winona C; Drabkin, Harold; Blake, Judith A; Smith, Barry; Wu, Cathy H

    2009-01-01

    Background The Protein Ontology (PRO) is designed as a formal and principled Open Biomedical Ontologies (OBO) Foundry ontology for proteins. The components of PRO extend from a classification of proteins on the basis of evolutionary relationships at the homeomorphic level to the representation of the multiple protein forms of a gene, including those resulting from alternative splicing, cleavage and/or post-translational modifications. Focusing specifically on the TGF-beta signaling proteins, we describe the building, curation, usage and dissemination of PRO. Results PRO is manually curated on the basis of PrePRO, an automatically generated file with content derived from standard protein data sources. Manual curation ensures that the treatment of the protein classes and the internal and external relationships conform to the PRO framework. The current release of PRO is based upon experimental data from mouse and human proteins wherein equivalent protein forms are represented by single terms. In addition to the PRO ontology, the annotation of PRO terms is released as a separate PRO association file, which contains, for each given PRO term, an annotation from the experimentally characterized sub-types as well as the corresponding database identifiers and sequence coordinates. The annotations are added in the form of relationship to other ontologies. Whenever possible, equivalent forms in other species are listed to facilitate cross-species comparison. Splice and allelic variants, gene fusion products and modified protein forms are all represented as entities in the ontology. Therefore, PRO provides for the representation of protein entities and a resource for describing the associated data. This makes PRO useful both for proteomics studies where isoforms and modified forms must be differentiated, and for studies of biological pathways, where representations need to take account of the different ways in which the cascade of events may depend on specific protein

  20. Evolution of Robustness to Protein Mistranslation by Accelerated Protein Turnover

    PubMed Central

    Farkas, Zoltán; Horvath, Peter; Bódi, Zoltán; Daraba, Andreea; Szamecz, Béla; Gut, Ivo; Bayes, Mónica; Santos, Manuel A. S.; Pál, Csaba

    2015-01-01

    Translational errors occur at high rates, and they influence organism viability and the onset of genetic diseases. To investigate how organisms mitigate the deleterious effects of protein synthesis errors during evolution, a mutant yeast strain was engineered to translate a codon ambiguously (mistranslation). It thereby overloads the protein quality-control pathways and disrupts cellular protein homeostasis. This strain was used to study the capacity of the yeast genome to compensate the deleterious effects of protein mistranslation. Laboratory evolutionary experiments revealed that fitness loss due to mistranslation can rapidly be mitigated. Genomic analysis demonstrated that adaptation was primarily mediated by large-scale chromosomal duplication and deletion events, suggesting that errors during protein synthesis promote the evolution of genome architecture. By altering the dosages of numerous, functionally related proteins simultaneously, these genetic changes introduced large phenotypic leaps that enabled rapid adaptation to mistranslation. Evolution increased the level of tolerance to mistranslation through acceleration of ubiquitin-proteasome–mediated protein degradation and protein synthesis. As a consequence of rapid elimination of erroneous protein products, evolution reduced the extent of toxic protein aggregation in mistranslating cells. However, there was a strong evolutionary trade-off between adaptation to mistranslation and survival upon starvation: the evolved lines showed fitness defects and impaired capacity to degrade mature ribosomes upon nutrient limitation. Moreover, as a response to an enhanced energy demand of accelerated protein turnover, the evolved lines exhibited increased glucose uptake by selective duplication of hexose transporter genes. We conclude that adjustment of proteome homeostasis to mistranslation evolves rapidly, but this adaptation has several side effects on cellular physiology. Our work also indicates that

  1. Protein models: The Grand Challenge of protein docking

    PubMed Central

    Anishchenko, Ivan; Kundrotas, Petras J.; Tuzikov, Alexander V.; Vakser, Ilya A.

    2016-01-01

    Characterization of life processes at the molecular level requires structural details of protein–protein interactions (PPIs). The number of experimentally determined protein structures accounts only for a fraction of known proteins. This gap has to be bridged by modeling, typically using experimentally determined structures as templates to model related proteins. The fraction of experimentally determined PPI structures is even smaller than that for the individual proteins, due to a larger number of interactions than the number of individual proteins, and a greater difficulty of crystallizing protein–protein complexes. The approaches to structural modeling of PPI (docking) often have to rely on modeled structures of the interactors, especially in the case of large PPI networks. Structures of modeled proteins are typically less accurate than the ones determined by X-ray crystallography or nuclear magnetic resonance. Thus the utility of approaches to dock these structures should be assessed by thorough benchmarking, specifically designed for protein models. To be credible, such benchmarking has to be based on carefully curated sets of structures with levels of distortion typical for modeled proteins. This article presents such a suite of models built for the benchmark set of the X-ray structures from the Dockground resource (http://dockground.bioinformatics.ku.edu) by a combination of homology modeling and Nudged Elastic Band method. For each monomer, six models were generated with predefined Cα root mean square deviation from the native structure (1, 2, . . ., 6 Å). The sets and the accompanying data provide a comprehensive resource for the development of docking methodology for modeled proteins. PMID:23934791

  2. Intracellular targeting with engineered proteins.

    PubMed

    Miersch, Shane; Sidhu, Sachdev S

    2016-01-01

    If the isolation, production, and clinical use of insulin marked the inception of the age of biologics as therapeutics, the convergence of molecular biology and combinatorial engineering techniques marked its coming of age. The first wave of recombinant protein-based drugs in the 1980s demonstrated emphatically that proteins could be engineered, formulated, and employed for clinical advantage. Yet despite the successes of protein-based drugs such as antibodies, enzymes, and cytokines, the druggable target space for biologics is currently restricted to targets outside the cell. Insofar as estimates place the number of proteins either secreted or with extracellular domains in the range of 8000 to 9000, this represents only one-third of the proteome and circumscribes the pathways that can be targeted for therapeutic intervention. Clearly, a major objective for this field to reach maturity is to access, interrogate, and modulate the majority of proteins found inside the cell. However, owing to the large size, complex architecture, and general cellular impermeability of existing protein-based drugs, this poses a daunting challenge. In recent years, though, advances on the two related fronts of protein engineering and drug delivery are beginning to bring this goal within reach. First, prompted by the restrictions that limit the applicability of antibodies, intense efforts have been applied to identifying and engineering smaller alternative protein scaffolds for the modulation of intracellular targets. In parallel, innovative solutions for delivering proteins to the intracellular space while maintaining their stability and functional activity have begun to yield successes. This review provides an overview of bioactive intrabodies and alternative protein scaffolds amenable to engineering for intracellular targeting and also outlines advances in protein engineering and formulation for delivery of functional proteins to the interior of the cell to achieve therapeutic action

  3. Scientist prepare Lysozyme Protein Crystal

    NASA Technical Reports Server (NTRS)

    1996-01-01

    Dan Carter and Charles Sisk center a Lysozyme Protein crystal grown aboard the USML-2 shuttle mission. Protein isolated from hen egg-white and functions as a bacteriostatic enzyme by degrading bacterial cell walls. First enzyme ever characterized by protein crystallography. It is used as an excellent model system for better understanding parameters involved in microgravity crystal growth experiments. The goal is to compare kinetic data from microgravity experiments with data from laboratory experiments to study the equilibrium.

  4. Protein Multifunctionality: Principles and Mechanisms

    PubMed Central

    Zaretsky, Joseph Z.; Wreschner, Daniel H.

    2008-01-01

    In the review, the nature of protein multifunctionality is analyzed. In the first part of the review the principles of structural/functional organization of protein are discussed. In the second part, the main mechanisms involved in development of multiple functions on a single gene product(s) are analyzed. The last part represents a number of examples showing that multifunctionality is a basic feature of biologically active proteins. PMID:21566747

  5. Containerless protein crystal growth method

    NASA Technical Reports Server (NTRS)

    Rhim, Won-Kyu; Chung, Sang K.

    1991-01-01

    A method of growing protein crystals from levitated drops is introduced and unique features of containerless approach in 1-g and micro-G laboratories are discussed. Electrostatic multidrop levitation system which is capable of simultaneous four drop levitation is described. A method of controlling protein saturation level in a programmed way is introduced and discussed. Finally, some of the unique features of containerless approach of protein crystal growth in space are discussed and summarized.

  6. Intracellular targeting with engineered proteins

    PubMed Central

    Miersch, Shane; Sidhu, Sachdev S.

    2016-01-01

    If the isolation, production, and clinical use of insulin marked the inception of the age of biologics as therapeutics, the convergence of molecular biology and combinatorial engineering techniques marked its coming of age. The first wave of recombinant protein-based drugs in the 1980s demonstrated emphatically that proteins could be engineered, formulated, and employed for clinical advantage. Yet despite the successes of protein-based drugs such as antibodies, enzymes, and cytokines, the druggable target space for biologics is currently restricted to targets outside the cell. Insofar as estimates place the number of proteins either secreted or with extracellular domains in the range of 8000 to 9000, this represents only one-third of the proteome and circumscribes the pathways that can be targeted for therapeutic intervention. Clearly, a major objective for this field to reach maturity is to access, interrogate, and modulate the majority of proteins found inside the cell. However, owing to the large size, complex architecture, and general cellular impermeability of existing protein-based drugs, this poses a daunting challenge. In recent years, though, advances on the two related fronts of protein engineering and drug delivery are beginning to bring this goal within reach. First, prompted by the restrictions that limit the applicability of antibodies, intense efforts have been applied to identifying and engineering smaller alternative protein scaffolds for the modulation of intracellular targets. In parallel, innovative solutions for delivering proteins to the intracellular space while maintaining their stability and functional activity have begun to yield successes. This review provides an overview of bioactive intrabodies and alternative protein scaffolds amenable to engineering for intracellular targeting and also outlines advances in protein engineering and formulation for delivery of functional proteins to the interior of the cell to achieve therapeutic action

  7. Developing algorithms for predicting protein-protein interactions of homology modeled proteins.

    SciTech Connect

    Martin, Shawn Bryan; Sale, Kenneth L.; Faulon, Jean-Loup Michel; Roe, Diana C.

    2006-01-01

    The goal of this project was to examine the protein-protein docking problem, especially as it relates to homology-based structures, identify the key bottlenecks in current software tools, and evaluate and prototype new algorithms that may be developed to improve these bottlenecks. This report describes the current challenges in the protein-protein docking problem: correctly predicting the binding site for the protein-protein interaction and correctly placing the sidechains. Two different and complementary approaches are taken that can help with the protein-protein docking problem. The first approach is to predict interaction sites prior to docking, and uses bioinformatics studies of protein-protein interactions to predict theses interaction site. The second approach is to improve validation of predicted complexes after docking, and uses an improved scoring function for evaluating proposed docked poses, incorporating a solvation term. This scoring function demonstrates significant improvement over current state-of-the art functions. Initial studies on both these approaches are promising, and argue for full development of these algorithms.

  8. The influence of protein coding sequences on protein folding rates of all-β proteins.

    PubMed

    Li, Rui Fang; Li, Hong

    2011-06-01

    It is currently believed that the protein folding rate is related to the protein structures and its amino acid sequence. However, few studies have been done on the problem that whether the protein folding rate is influenced by its corresponding mRNA sequence. In this paper, we analyzed the possible relationship between the protein folding rates and the corresponding mRNA sequences. The content of guanine and cytosine (GC content) of palindromes in protein coding sequence was introduced as a new parameter and added in the Gromiha's model of predicting protein folding rates to inspect its effect in protein folding process. The multiple linear regression analysis and jack-knife test show that the new parameter is significant. The linear correlation coefficient between the experimental and the predicted values of the protein folding rates increased significantly from 0.96 to 0.99, and the population variance decreased from 0.50 to 0.24 compared with Gromiha's results. The results show that the GC content of palindromes in the corresponding protein coding sequence really influences the protein folding rate. Further analysis indicates that this kind of effect mostly comes from the synonymous codon usage and from the information of palindrome structure itself, but not from the translation information from codons to amino acids. PMID:21613670

  9. Identification of the First Prokaryotic Collagen Sequence Motif That Mediates Binding to Human Collagen Receptors, Integrins α2β1 and α11β1*

    PubMed Central

    Caswell, Clayton C.; Barczyk, Malgorzata; Keene, Douglas R.; Lukomska, Ewa; Gullberg, Donald E.; Lukomski, Slawomir

    2008-01-01

    Many pathogenic bacteria interact with human integrins to enter host cells and to augment host colonization. Group A Streptococcus (GAS) employs molecular mimicry by direct interactions between the cell surface streptococcal collagen-like protein-1 (Scl1) and the human collagen receptor, integrin α2β1. The collagen-like (CL) region of the Scl1 protein mediates integrin-binding, although, the integrin binding motif was not defined. Here, we used molecular cloning and site-directed mutagenesis to identify the GLPGER sequence as the α2β1 and the α11β1 binding motif. Electron microscopy experiments mapped binding sites of the recombinant α2-integrin-inserted domain to the GLPGER motif of the recombinant Scl (rScl) protein. rScl proteins and a synthetic peptide harboring the GLPGER motif mediated the attachment of C2C12-α2 + myoblasts expressing the α2β1 integrin as the sole collagen receptor. The C2C12-α11 + myoblasts expressing the α11β1 integrin also attached to GLPGER-harboring rScl proteins. Furthermore, the C2C12-α11 + cells attached to rScl1 more efficiently than C2C12-α2 + cells, suggesting that the α11β1 integrin may have a higher binding affinity for the GLPGER sequence. Human endothelial cells and dermal fibroblasts adhered to rScl proteins, indicating that multiple cell types may recognize and bind the Scl proteins via their collagen receptors. This work is a stepping stone toward defining the utilization of collagen receptors by microbial collagen-like proteins that are expressed by pathogenic bacteria. PMID:18990704

  10. Harvesting blood proteins from grain.

    PubMed Central

    Robinson, A

    1995-01-01

    A multidisciplinary team of researchers at the University of Ottawa has expressed a human blood protein, granulocyte-macrophage colony stimulating factor, in tobacco seeds as part of a series of experiments whose ultimate goal is to express human blood proteins in cereal crops. Success in these experiments may lead to the development of a new, relatively inexpensive and ready supply of these proteins from a biologic source that is generally recognized as safe. The team is also studying the possibility of expressing in seeds proteins that may be used as vaccines against infectious diseases. Images p428-a Fig. 1 PMID:7634220

  11. Protein phosphorylation in stomatal movement

    PubMed Central

    Zhang, Tong; Chen, Sixue; Harmon, Alice C

    2014-01-01

    As research progresses on how guard cells perceive and transduce environmental cues to regulate stomatal movement, plant biologists are discovering key roles of protein phosphorylation. Early research efforts focused on characterization of ion channels and transporters in guard cell hormonal signaling. Subsequent genetic studies identified mutants of kinases and phosphatases that are defective in regulating guard cell ion channel activities, and recently proteins regulated by phosphorylation have been identified. Here we review the essential role of protein phosphorylation in ABA-induced stomatal closure and in blue light-induced stomatal opening. We also highlight evidence for the cross-talk between different pathways, which is mediated by protein phosphorylation. PMID:25482764

  12. Conformational changes of adsorbed proteins

    NASA Astrophysics Data System (ADS)

    Allen, Scott

    2005-03-01

    The adsorption of bovine serum albumin (BSA) and pepsin to gold surfaces has been studied using surface plasmon resonance (SPR). Proteins are adsorbed from solution onto a gold surface and changes in the conformation of the adsorbed proteins are induced by changing the buffer solution. We selected pH and ionic strength values for the buffer solutions that are known from our circular dichroism measurements to cause conformational changes of the proteins in bulk solution. We find that for both BSA and pepsin the changes in conformation are impeded by the interaction of the protein with the gold surface.

  13. Airborne concentrations of peanut protein.

    PubMed

    Johnson, Rodney M; Barnes, Charles S

    2013-01-01

    Food allergy to peanut is a significant health problem, and there are reported allergic reactions to peanuts despite not eating or having physical contact with peanuts. It is presumed that an allergic reaction may have occurred from inhalation of airborne peanut allergens. The purpose of this study was to detect the possible concentrations of airborne peanut proteins for various preparations and during specific activities. Separate Ara h 1 and Ara h 2 monoclonal enzyme-linked immunosorbent assays and a polyclonal sandwich enzyme immunoassay for peanuts were used to detect the amount of airborne peanut protein collected using a Spincon Omni 3000 air collector (Sceptor Industries, Inc., Kansas City, MO) under different peanut preparation methods and situations. Air samples were measured for multiple peanut preparations and scenarios. Detectable amounts of airborne peanut protein were measured using a whole peanut immunoassay when removing the shells of roasted peanut. No airborne peanut allergen (Ara h 1 or Ara h 2) or whole peanut protein above the LLD was measured in any of the other peanut preparation collections. Ara h 1, Ara h 2, and polyclonal peanut proteins were detected from water used to boil peanuts. Small amounts of airborne peanut protein were detected in the scenario of removing shells from roasted peanuts; however, Ara h 1 and Ara h 2 proteins were unable to be consistently detected. Although airborne peanut proteins were detected, the concentration of airborne peanut protein that is necessary to elicit a clinical allergic reaction is unknown. PMID:23406937

  14. Cellular senescence and protein degradation

    PubMed Central

    Deschênes-Simard, Xavier; Lessard, Frédéric; Gaumont-Leclerc, Marie-France; Bardeesy, Nabeel; Ferbeyre, Gerardo

    2014-01-01

    Autophagy and the ubiquitin–proteasome pathway (UPP) are the major protein degradation systems in eukaryotic cells. Whereas the former mediate a bulk nonspecific degradation, the UPP allows a rapid degradation of specific proteins. Both systems have been shown to play a role in tumorigenesis, and the interest in developing therapeutic agents inhibiting protein degradation is steadily growing. However, emerging data point to a critical role for autophagy in cellular senescence, an established tumor suppressor mechanism. Recently, a selective protein degradation process mediated by the UPP was also shown to contribute to the senescence phenotype. This process is tightly regulated by E3 ubiquitin ligases, deubiquitinases, and several post-translational modifications of target proteins. Illustrating the complexity of UPP, more than 600 human genes have been shown to encode E3 ubiquitin ligases, a number which exceeds that of the protein kinases. Nevertheless, our knowledge of proteasome-dependent protein degradation as a regulated process in cellular contexts such as cancer and senescence remains very limited. Here we discuss the implications of protein degradation in senescence and attempt to relate this function to the protein degradation pattern observed in cancer cells. PMID:24866342

  15. Amyloidogenesis of Natively Unfolded Proteins

    PubMed Central

    Uversky, Vladimir N.

    2009-01-01

    Aggregation and subsequent development of protein deposition diseases originate from conformational changes in corresponding amyloidogenic proteins. The accumulated data support the model where protein fibrillogenesis proceeds via the formation of a relatively unfolded amyloidogenic conformation, which shares many structural properties with the pre-molten globule state, a partially folded intermediate first found during the equilibrium and kinetic (un)folding studies of several globular proteins and later described as one of the structural forms of natively unfolded proteins. The flexibility of this structural form is essential for the conformational rearrangements driving the formation of the core cross-beta structure of the amyloid fibril. Obviously, molecular mechanisms describing amyloidogenesis of ordered and natively unfolded proteins are different. For ordered protein to fibrillate, its unique and rigid structure has to be destabilized and partially unfolded. On the other hand, fibrillogenesis of a natively unfolded protein involves the formation of partially folded conformation; i.e., partial folding rather than unfolding. In this review recent findings are surveyed to illustrate some unique features of the natively unfolded proteins amyloidogenesis. PMID:18537543

  16. [Protein toxins of Staphylococcus aureus].

    PubMed

    Shamsutdinov, A F; Tiurin, Iu A

    2014-01-01

    Main scientific-research studies regarding protein bacterial toxins of the most widespread bacteria that belong to Staphylococcus spp. genus and in particular the most pathogenic species for humans--Staphylococcus aureus, are analyzed. Structural and biological properties of protein toxins that have received the name of staphylococcus pyrogenic toxins (PTSAg) are presented. Data regarding genetic regulation of secretion and synthesis of these toxins and 3 main regulatory genetic systems (agr--accessory gene regulator, xpr--extracellular protein regulator, sar--staphylococcal accessory regulator) that coordinate synthesis of the most important protein toxins and enzymes for virulence of S. aureus, are presented. PMID:25051707

  17. Therapeutic proteins: A to Z.

    PubMed

    Ozgur, Aykut; Tutar, Yusuf

    2013-12-01

    In recent years, therapeutic proteins have become an important growing class of drugs in the pharmaceutics industry. The development of recombinant DNA technology has caused to appreciation of therapeutic value of many proteins and peptides in medicine. Currently, approximately 100 therapeutic proteins obtained approval from Food and Drug Administration (FDA) and they are widely used in the treatment of various diseases such as cancer, diabetes, anemia and infections. This paper will summarize the production processes, pharmaceuticals and physicochemical properties and important classes of therapeutic proteins with their potential use in clinical applications. PMID:24261980

  18. Membrane Protein Assembly into Nanodiscs

    PubMed Central

    Bayburt, Timothy H.; Sligar, Stephen G.

    2016-01-01

    Nanodiscs are soluble nanoscale phospholipid bilayers which can self-assemble integral membrane proteins for biophysical, enzymatic or structural investigations. This means for rendering membrane proteins soluble at the single molecule level offers advantages over liposomes or detergent micelles in terms of size, stability, ability to add genetically modifiable features to the Nanodisc structure and ready access to both sides of the phospholipid bilayer domain. Thus the Nanodisc system provides a novel platform for understanding membrane protein function. We provide an overview of the Nanodisc approach and document through several examples many of the applications to the study of the structure and function of integral membrane proteins. PMID:19836392

  19. Structure Prediction of Membrane Proteins

    NASA Astrophysics Data System (ADS)

    Hu, Xiche

    Membrane proteins play a central role in many cellular and physiological processes. It is estimated that integral membrane proteins make up about 20-30% of the proteome (Krogh et al., 2001b; Stevens and Arkin, 2000; von Heijne, 1999). They are essential mediators of material and information transfer across cell membranes. Their functions include active and passive transport of molecules into and out of cells and organelles; transduction of energy among various forms (light, electrical, and chemical energy); as well as reception and transduction of chemical and electrical signals across membranes (Avdonin, 2005; Bockaert et al., 2002; Pahl, 1999; Rehling et al., 2004; Stack et al., 1995). Identifying these transmembrane (TM) proteins and deciphering their molecular mechanisms, then, is of great importance, particularly as applied to biomedicine. Membrane proteins are the targets of a large number of pharmacologically and toxicologically active substances, and are directly involved in their uptake, metabolism, and clearance (Bettler et al., 1998; Cohen, 2002; Heusser and Jardieu, 1997; Tibes et al., 2005; Xu et al., 2005). Despite the importance of membrane proteins, the knowledge of their high-resolution structures and mechanisms of action has lagged far behind in comparison to that of water-soluble proteins: less than 1% of all three-dimensional structures deposited in the Protein Data Bank are of membrane proteins. This unfortunate disparity stems from difficulties in overexpression and the crystallization of membrane proteins (Grisshammer and Tate, 1995; Michel, 1991).

  20. Molecular dynamics of membrane proteins.

    SciTech Connect

    Woolf, Thomas B.; Crozier, Paul Stewart; Stevens, Mark Jackson

    2004-10-01

    Understanding the dynamics of the membrane protein rhodopsin will have broad implications for other membrane proteins and cellular signaling processes. Rhodopsin (Rho) is a light activated G-protein coupled receptor (GPCR). When activated by ligands, GPCRs bind and activate G-proteins residing within the cell and begin a signaling cascade that results in the cell's response to external stimuli. More than 50% of all current drugs are targeted toward G-proteins. Rho is the prototypical member of the class A GPCR superfamily. Understanding the activation of Rho and its interaction with its Gprotein can therefore lead to a wider understanding of the mechanisms of GPCR activation and G-protein activation. Understanding the dark to light transition of Rho is fully analogous to the general ligand binding and activation problem for GPCRs. This transition is dependent on the lipid environment. The effect of lipids on membrane protein activity in general has had little attention, but evidence is beginning to show a significant role for lipids in membrane protein activity. Using the LAMMPS program and simulation methods benchmarked under the IBIG program, we perform a variety of allatom molecular dynamics simulations of membrane proteins.

  1. Nanotube-assisted protein deactivation

    NASA Astrophysics Data System (ADS)

    Joshi, Amit; Punyani, Supriya; Bale, Shyam Sundhar; Yang, Hoichang; Borca-Tasciuc, Theodorian; Kane, Ravi S.

    2008-01-01

    Conjugating proteins onto carbon nanotubes has numerous applications in biosensing, imaging and cellular delivery. However, remotely controlling the activity of proteins in these conjugates has never been demonstrated. Here we show that upon near-infrared irradiation, carbon nanotubes mediate the selective deactivation of proteins in situ by photochemical effects. We designed nanotube-peptide conjugates to selectively destroy the anthrax toxin, and also optically transparent coatings that can self-clean following either visible or near-infrared irradiation. Nanotube-assisted protein deactivation may be broadly applicable to the selective destruction of pathogens and cells, and will have applications ranging from antifouling coatings to functional proteomics.

  2. Nucleation precursors in protein crystallization

    PubMed Central

    Vekilov, Peter G.; Vorontsova, Maria A.

    2014-01-01

    Protein crystal nucleation is a central problem in biological crystallography and other areas of science, technology and medicine. Recent studies have demonstrated that protein crystal nuclei form within crucial precursors. Here, methods of detection and characterization of the precursors are reviewed: dynamic light scattering, atomic force microscopy and Brownian microscopy. Data for several proteins provided by these methods have demonstrated that the nucleation precursors are clusters consisting of protein-dense liquid, which are metastable with respect to the host protein solution. The clusters are several hundred nanometres in size, the cluster population occupies from 10−7 to 10−3 of the solution volume, and their properties in solutions supersaturated with respect to crystals are similar to those in homogeneous, i.e. undersaturated, solutions. The clusters exist owing to the conformation flexibility of the protein molecules, leading to exposure of hydrophobic surfaces and enhanced intermolecular binding. These results indicate that protein conformational flexibility might be the mechanism behind the metastable mesoscopic clusters and crystal nucleation. Investigations of the cluster properties are still in their infancy. Results on direct imaging of cluster behaviors and characterization of cluster mechanisms with a variety of proteins will soon lead to major breakthroughs in protein biophysics. PMID:24598910

  3. High throughput protein production screening

    DOEpatents

    Beernink, Peter T.; Coleman, Matthew A.; Segelke, Brent W.

    2009-09-08

    Methods, compositions, and kits for the cell-free production and analysis of proteins are provided. The invention allows for the production of proteins from prokaryotic sequences or eukaryotic sequences, including human cDNAs using PCR and IVT methods and detecting the proteins through fluorescence or immunoblot techniques. This invention can be used to identify optimized PCR and WT conditions, codon usages and mutations. The methods are readily automated and can be used for high throughput analysis of protein expression levels, interactions, and functional states.

  4. Protein phosphorylation in stomatal movement.

    PubMed

    Zhang, Tong; Chen, Sixue; Harmon, Alice C

    2014-01-01

    As research progresses on how guard cells perceive and transduce environmental cues to regulate stomatal movement, plant biologists are discovering key roles of protein phosphorylation. Early research efforts focused on characterization of ion channels and transporters in guard cell hormonal signaling. Subsequent genetic studies identified mutants of kinases and phosphatases that are defective in regulating guard cell ion channel activities, and recently proteins regulated by phosphorylation have been identified. Here we review the essential role of protein phosphorylation in ABA-induced stomatal closure and in blue light-induced stomatal opening. We also highlight evidence for the cross-talk between different pathways, which is mediated by protein phosphorylation. PMID:25482764

  5. Immunoprofiling Using NAPPA Protein Microarrays

    PubMed Central

    Sibani, Sahar; LaBaer, Joshua

    2012-01-01

    Protein microarrays provide an efficient method to immunoprofile patients in an effort to rapidly identify disease immunosignatures. The validity of using autoantibodies in diagnosis has been demonstrated in type 1 diabetes, rheumatoid arthritis, and systemic lupus, and is now being strongly considered in cancer. Several types of protein microarrays exist including antibody and antigen arrays. In this chapter, we describe the immunoprofiling application for one type of antigen array called NAPPA (nucleic acids programmable protein array). We provide a guideline for setting up the screening study and designing protein arrays to maximize the likelihood of obtaining quality data. PMID:21370064

  6. The Protein 4.1 family: hub proteins in animals for organizing membrane proteins.

    PubMed

    Baines, Anthony J; Lu, Hui-Chun; Bennett, Pauline M

    2014-02-01

    Proteins of the 4.1 family are characteristic of eumetazoan organisms. Invertebrates contain single 4.1 genes and the Drosophila model suggests that 4.1 is essential for animal life. Vertebrates have four paralogues, known as 4.1R, 4.1N, 4.1G and 4.1B, which are additionally duplicated in the ray-finned fish. Protein 4.1R was the first to be discovered: it is a major mammalian erythrocyte cytoskeletal protein, essential to the mechanochemical properties of red cell membranes because it promotes the interaction between spectrin and actin in the membrane cytoskeleton. 4.1R also binds certain phospholipids and is required for the stable cell surface accumulation of a number of erythrocyte transmembrane proteins that span multiple functional classes; these include cell adhesion molecules, transporters and a chemokine receptor. The vertebrate 4.1 proteins are expressed in most tissues, and they are required for the correct cell surface accumulation of a very wide variety of membrane proteins including G-Protein coupled receptors, voltage-gated and ligand-gated channels, as well as the classes identified in erythrocytes. Indeed, such large numbers of protein interactions have been mapped for mammalian 4.1 proteins, most especially 4.1R, that it appears that they can act as hubs for membrane protein organization. The range of critical interactions of 4.1 proteins is reflected in disease relationships that include hereditary anaemias, tumour suppression, control of heartbeat and nervous system function. The 4.1 proteins are defined by their domain structure: apart from the spectrin/actin-binding domain they have FERM and FERM-adjacent domains and a unique C-terminal domain. Both the FERM and C-terminal domains can bind transmembrane proteins, thus they have the potential to be cross-linkers for membrane proteins. The activity of the FERM domain is subject to multiple modes of regulation via binding of regulatory ligands, phosphorylation of the FERM associated domain and

  7. Proteins interacting with Membranes: Protein Sorting and Membrane Shaping

    NASA Astrophysics Data System (ADS)

    Callan-Jones, Andrew

    2015-03-01

    Membrane-bound transport in cells requires generating membrane curvature. In addition, transport is selective, in order to establish spatial gradients of membrane components in the cell. The mechanisms underlying cell membrane shaping by proteins and the influence of curvature on membrane composition are active areas of study in cell biophysics. In vitro approaches using Giant Unilamellar Vesicles (GUVs) are a useful tool to identify the physical mechanisms that drive sorting of membrane components and membrane shape change by proteins. I will present recent work on the curvature sensing and generation of IRSp53, a protein belonging to the BAR family, whose members, sharing a banana-shaped backbone, are involved in endocytosis. Pulling membrane tubes with 10-100 nm radii from GUVs containing encapsulated IRSp53 have, unexpectedly, revealed a non-monotonic dependence of the protein concentration on the tube as a function of curvature. Experiments also show that bound proteins alter the tube mechanics and that protein phase separation along the tube occurs at low tensions. I will present accompanying theoretical work that can explain these findings based on the competition between the protein's intrinsic curvature and the effective rigidity of a membrane-protein patch.

  8. Water sorption by proteins: milk and whey proteins.

    PubMed

    Kinsella, J E; Fox, P F

    1986-01-01

    The content and physical state of water in foods influence their physical, chemical, quality, safety, and functional behavior. Information concerning the sorption behavior of dairy proteins, in the water activity (Aw) range 0 to 0.9, is collated in this paper. The sorption behavior of proteins in general, the kinetics of absorption, factors affecting water binding, the phenomenon of desorption hysteresis, and the chemical and physical nature of water/protein interactions are reviewed in general terms. This is followed by a discussion of thermodynamic aspects of sorption phenomena and the adequacy of the various equations for describing sorption isotherms of proteins. After a discussion of the methods available for measuring sorption by milk proteins, the sorption behavior of various milk protein preparations, i.e., nonfat dry milk, whey proteins, caseins, and milk powders is summarized. Finally, the water activity of cheese and its relationship to solute mobility and solvent water are discussed. Some of the unique features of protein behavior, i.e., conformational changes, swelling, and solubilization are cited as possible sources of disparities between various reports. PMID:3527564

  9. Website on Protein Interaction and Protein Structure Related Work

    NASA Technical Reports Server (NTRS)

    Samanta, Manoj; Liang, Shoudan; Biegel, Bryan (Technical Monitor)

    2003-01-01

    In today's world, three seemingly diverse fields - computer information technology, nanotechnology and biotechnology are joining forces to enlarge our scientific knowledge and solve complex technological problems. Our group is dedicated to conduct theoretical research exploring the challenges in this area. The major areas of research include: 1) Yeast Protein Interactions; 2) Protein Structures; and 3) Current Transport through Small Molecules.

  10. Non-enzymatic protein acetylation detected by NAPPA protein arrays*

    PubMed Central

    Olia, Adam S.; Barker, Kristi; McCullough, Cheryl E.; Tang, Hsin-Yao; Speicher, David W.; Qiu, Ji; LaBaer, Joshua; Marmorstein, Ronen

    2015-01-01

    Acetylation is a post-translational modification that occurs on thousands of proteins located in many cellular organelles. This process mediates many protein functions and modulates diverse biological processes. In mammalian cells, where acetyl-CoA is the primary acetyl donor, acetylation in the mitochondria is thought to occur by chemical means due to the relatively high concentration of acetyl-CoA located in this organelle. In contrast, acetylation outside of the mitochondria is thought to be mediated predominantly by acetyltransferase enzymes. Here we address the possibility that non-enzymatic chemical acetylation outside of the mitochondria may be more common than previously appreciated. We employed the Nucleic Acid Programmable Protein Array platform to perform an unbiased screen for human proteins that undergo chemical acetylation, which resulted in the identification of a multitude of proteins with diverse functions and cellular localization. Mass spectrometry analysis revealed that basic residues typically precede the acetylated lysine in the −7 to −3 position, and we show by mutagenesis that these basic residues contribute to chemical acetylation capacity. We propose that these basic residues lower the pKa of the substrate lysine for efficient chemical acetylation. Many of the identified proteins reside outside of the mitochondria, and have been previously demonstrated to be acetylated in vivo. As such, our studies demonstrate that chemical acetylation occurs more broadly throughout the eukaryotic cell than previously appreciated, and suggests that this post-translational protein modification may have more diverse roles in protein function and pathway regulation. PMID:26083674

  11. MYCOPLASMA GENITALIUM PROTEIN RESEMBLING THE MYCOPLASMA PNEUMONIAE ATTACHMENT PROTEIN

    EPA Science Inventory

    In previous studies with hyperimmune rabbit sera and monoclonal antibodies against P1 protein of M. pneumoniae, we obtained evidence of a shared antigenic determinant with a single protein of M. genitalium. ecause of biological and morphological similarities between these two hum...

  12. Eukaryotic LYR Proteins Interact with Mitochondrial Protein Complexes.

    PubMed

    Angerer, Heike

    2015-01-01

    In eukaryotic cells, mitochondria host ancient essential bioenergetic and biosynthetic pathways. LYR (leucine/tyrosine/arginine) motif proteins (LYRMs) of the Complex1_LYR-like superfamily interact with protein complexes of bacterial origin. Many LYR proteins function as extra subunits (LYRM3 and LYRM6) or novel assembly factors (LYRM7, LYRM8, ACN9 and FMC1) of the oxidative phosphorylation (OXPHOS) core complexes. Structural insights into complex I accessory subunits LYRM6 and LYRM3 have been provided by analyses of EM and X-ray structures of complex I from bovine and the yeast Yarrowia lipolytica, respectively. Combined structural and biochemical studies revealed that LYRM6 resides at the matrix arm close to the ubiquinone reduction site. For LYRM3, a position at the distal proton-pumping membrane arm facing the matrix space is suggested. Both LYRMs are supposed to anchor an acyl-carrier protein (ACPM) independently to complex I. The function of this duplicated protein interaction of ACPM with respiratory complex I is still unknown. Analysis of protein-protein interaction screens, genetic analyses and predicted multi-domain LYRMs offer further clues on an interaction network and adaptor-like function of LYR proteins in mitochondria. PMID:25686363

  13. Including Ligand Induced Protein Flexibility into Protein Tunnel Prediction

    PubMed Central

    Kingsley, Laura J.; Lill, Markus A.

    2014-01-01

    In proteins with buried active sites, understanding how ligands migrate through the tunnels that connect the exterior of the protein to the active site can shed light on substrate specificity and enzyme function. A growing body of evidence highlights the importance of protein flexibility in the binding site upon ligand binding; however, the influence of protein flexibility throughout the body of the protein during ligand entry and egress is much less characterized. We have developed a novel tunnel prediction and evaluation method named IterTunnel, which includes the influence of ligand-induced protein flexibility, guarantees ligand egress, and provides detailed free energy information as the ligand proceeds along the egress route. IterTunnel combines geometric tunnel prediction with steered MD in an iterative process to identify tunnels that open as a result of ligand migration and calculates the potential of mean force (PMF) of ligand egress through a given tunnel. Applying this new method to cytochrome P450 2B6 (CYP2B6), we demonstrate the influence of protein flexibility on the shape and accessibility of tunnels. More importantly, we demonstrate that the ligand itself, while traversing through a tunnel, can reshape tunnels due to its interaction with the protein. This process results in the exposure of new tunnels and the closure of pre-existing tunnels as the ligand migrates from the active site. PMID:25043499

  14. Ribo-Proteomics Approach to Profile RNA-Protein and Protein-Protein Interaction Networks.

    PubMed

    Yeh, Hsin-Sung; Chang, Jae-Woong; Yong, Jeongsik

    2016-01-01

    Characterizing protein-protein and protein-RNA interaction networks is a fundamental step to understanding the function of an RNA-binding protein. In many cases, these interactions are transient and highly dynamic. Therefore, capturing stable as well as transient interactions in living cells for the identification of protein-binding partners and the mapping of RNA-binding sequences is key to a successful establishment of the molecular interaction network. In this chapter, we will describe a method for capturing the molecular interactions in living cells using formaldehyde as a crosslinker and enriching a specific RNA-protein complex from cell extracts followed by mass spectrometry and Next-Gen sequencing analyses. PMID:26965265

  15. Truly Absorbed Microbial Protein Synthesis, Rumen Bypass Protein, Endogenous Protein, and Total Metabolizable Protein from Starchy and Protein-Rich Raw Materials: Model Comparison and Predictions.

    PubMed

    Parand, Ehsan; Vakili, Alireza; Mesgaran, Mohsen Danesh; van Duinkerken, Gert; Yu, Peiqiang

    2015-07-29

    This study was carried out to measure truly absorbed microbial protein synthesis, rumen bypass protein, and endogenous protein loss, as well as total metabolizable protein, from starchy and protein-rich raw feed materials with model comparisons. Predictions by the DVE2010 system as a more mechanistic model were compared with those of two other models, DVE1994 and NRC-2001, that are frequently used in common international feeding practice. DVE1994 predictions for intestinally digestible rumen undegradable protein (ARUP) for starchy concentrates were higher (27 vs 18 g/kg DM, p < 0.05, SEM = 1.2) than predictions by the NRC-2001, whereas there was no difference in predictions for ARUP from protein concentrates among the three models. DVE2010 and NRC-2001 had highest estimations of intestinally digestible microbial protein for starchy (92 g/kg DM in DVE2010 vs 46 g/kg DM in NRC-2001 and 67 g/kg DM in DVE1994, p < 0.05 SEM = 4) and protein concentrates (69 g/kg DM in NRC-2001 vs 31 g/kg DM in DVE1994 and 49 g/kg DM in DVE2010, p < 0.05 SEM = 4), respectively. Potential protein supplies predicted by tested models from starchy and protein concentrates are widely different, and comparable direct measurements are needed to evaluate the actual ability of different models to predict the potential protein supply to dairy cows from different feedstuffs. PMID:26118653

  16. PPIM: A Protein-Protein Interaction Database for Maize.

    PubMed

    Zhu, Guanghui; Wu, Aibo; Xu, Xin-Jian; Xiao, Pei-Pei; Lu, Le; Liu, Jingdong; Cao, Yongwei; Chen, Luonan; Wu, Jun; Zhao, Xing-Ming

    2016-02-01

    Maize (Zea mays) is one of the most important crops worldwide. To understand the biological processes underlying various traits of the crop (e.g. yield and response to stress), a detailed protein-protein interaction (PPI) network is highly demanded. Unfortunately, there are very few such PPIs available in the literature. Therefore, in this work, we present the Protein-Protein Interaction Database for Maize (PPIM), which covers 2,762,560 interactions among 14,000 proteins. The PPIM contains not only accurately predicted PPIs but also those molecular interactions collected from the literature. The database is freely available at http://comp-sysbio.org/ppim with a user-friendly powerful interface. We believe that the PPIM resource can help biologists better understand the maize crop. PMID:26620522

  17. Prediction and redesign of protein-protein interactions.

    PubMed

    Lua, Rhonald C; Marciano, David C; Katsonis, Panagiotis; Adikesavan, Anbu K; Wilkins, Angela D; Lichtarge, Olivier

    2014-01-01

    Understanding the molecular basis of protein function remains a central goal of biology, with the hope to elucidate the role of human genes in health and in disease, and to rationally design therapies through targeted molecular perturbations. We review here some of the computational techniques and resources available for characterizing a critical aspect of protein function - those mediated by protein-protein interactions (PPI). We describe several applications and recent successes of the Evolutionary Trace (ET) in identifying molecular events and shapes that underlie protein function and specificity in both eukaryotes and prokaryotes. ET is a part of analytical approaches based on the successes and failures of evolution that enable the rational control of PPI. PMID:24878423

  18. Control of repeat protein curvature by computational protein design

    PubMed Central

    Park, Keunwan; Shen, Betty W.; Parmeggiani, Fabio; Huang, Po-Ssu; Stoddard, Barry L.; Baker, David

    2014-01-01

    Shape complementarity is an important component of molecular recognition, and the ability to precisely adjust the shape of a binding scaffold to match a target of interest would greatly facilitate the creation of high affinity protein reagents and therapeutics. Here we describe a general approach to control the shape of the binding surface on repeat protein scaffolds, and apply it to leucine rich repeat proteins. First, a set of self-compatible building block modules are designed that when polymerized each generate surfaces with unique but constant curvatures. Second, a set of junction modules that connect the different building blocks are designed. Finally, new proteins with custom designed shapes are generated by appropriately combining building block and junction modules. Crystal structures of the designs illustrate the power of the approach in controlling repeat protein curvature. PMID:25580576

  19. Protein-protein interaction networks (PPI) and complex diseases

    PubMed Central

    Safari-Alighiarloo, Nahid; Taghizadeh, Mohammad; Rezaei-Tavirani, Mostafa; Goliaei, Bahram

    2014-01-01

    The physical interaction of proteins which lead to compiling them into large densely connected networks is a noticeable subject to investigation. Protein interaction networks are useful because of making basic scientific abstraction and improving biological and biomedical applications. Based on principle roles of proteins in biological function, their interactions determine molecular and cellular mechanisms, which control healthy and diseased states in organisms. Therefore, such networks facilitate the understanding of pathogenic (and physiologic) mechanisms that trigger the onset and progression of diseases. Consequently, this knowledge can be translated into effective diagnostic and therapeutic strategies. Furthermore, the results of several studies have proved that the structure and dynamics of protein networks are disturbed in complex diseases such as cancer and autoimmune disorders. Based on such relationship, a novel paradigm is suggested in order to confirm that the protein interaction networks can be the target of therapy for treatment of complex multi-genic diseases rather than individual molecules with disrespect the network. PMID:25436094

  20. Information-driven structural modelling of protein-protein interactions.

    PubMed

    Rodrigues, João P G L M; Karaca, Ezgi; Bonvin, Alexandre M J J

    2015-01-01

    Protein-protein docking aims at predicting the three-dimensional structure of a protein complex starting from the free forms of the individual partners. As assessed in the CAPRI community-wide experiment, the most successful docking algorithms combine pure laws of physics with information derived from various experimental or bioinformatics sources. Of these so-called "information-driven" approaches, HADDOCK stands out as one of the most successful representatives. In this chapter, we briefly summarize which experimental information can be used to drive the docking prediction in HADDOCK, and then focus on the docking protocol itself. We discuss and illustrate with a tutorial example a "classical" protein-protein docking prediction, as well as more recent developments for modelling multi-body systems and large conformational changes. PMID:25330973