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Sample records for actin-binding protein cofilin

  1. Mechanosensitive kinetic preference of actin-binding protein to actin filament

    NASA Astrophysics Data System (ADS)

    Inoue, Yasuhiro; Adachi, Taiji

    2016-04-01

    The kinetic preference of actin-binding proteins to actin filaments is altered by external forces on the filament. Such an altered kinetic preference is largely responsible for remodeling the actin cytoskeletal structure in response to intracellular forces. During remodeling, actin-binding proteins and actin filaments interact under isothermal conditions, because the cells are homeostatic. In such a temperature homeostatic state, we can rigorously and thermodynamically link the chemical potential of actin-binding proteins to stresses on the actin filaments. From this relationship, we can construct a physical model that explains the force-dependent kinetic preference of actin-binding proteins to actin filaments. To confirm the model, we have analyzed the mechanosensitive alternation of the kinetic preference of Arp2/3 and cofilin to actin filaments. We show that this model captures the qualitative responses of these actin-binding proteins to the forces, as observed experimentally. Moreover, our theoretical results demonstrate that, depending on the structural parameters of the binding region, actin-binding proteins can show different kinetic responses even to the same mechanical signal tension, in which the double-helix nature of the actin filament also plays a critical role in a stretch-twist coupling of the filament.

  2. Actin binding proteins, spermatid transport and spermiation*

    PubMed Central

    Qian, Xiaojing; Mruk, Dolores D.; Cheng, Yan-Ho; Tang, Elizabeth I.; Han, Daishu; Lee, Will M.; Wong, Elissa W. P.; Cheng, C. Yan

    2014-01-01

    The transport of germ cells across the seminiferous epithelium is composed of a series of cellular events during the epithelial cycle essential to the completion of spermatogenesis. Without the timely transport of spermatids during spermiogenesis, spermatozoa that are transformed from step 19 spermatids in the rat testis fail to reach the luminal edge of the apical compartment and enter the tubule lumen at spermiation, thereby entering the epididymis for further maturation. Step 19 spermatids and/or sperms that remain in the epithelium will be removed by the Sertoli cell via phagocytosis to form phagosomes and be degraded by lysosomes, leading to subfertility and/or infertility. However, the biology of spermatid transport, in particular the final events that lead to spermiation remain elusive. Based on recent data in the field, we critically evaluate the biology of spermiation herein by focusing on the actin binding proteins (ABPs) that regulate the organization of actin microfilaments at the Sertoli-spermatid interface, which is crucial for spermatid transport during this event. The hypothesis we put forth herein also highlights some specific areas of research that can be pursued by investigators in the years to come. PMID:24735648

  3. The actin binding protein adseverin regulates osteoclastogenesis.

    PubMed

    Hassanpour, Siavash; Jiang, Hongwei; Wang, Yongqiang; Kuiper, Johannes W P; Glogauer, Michael

    2014-01-01

    Adseverin (Ads), a member of the Gelsolin superfamily of actin binding proteins, regulates the actin cytoskeleton architecture by severing and capping existing filamentous actin (F-actin) strands and nucleating the assembly of new F-actin filaments. Ads has been implicated in cellular secretion, exocytosis and has also been shown to regulate chondrogenesis and megakaryoblastic leukemia cell differentiation. Here we report for the first time that Ads is involved in regulating osteoclastogenesis (OCG). Ads is induced during OCG downstream of RANK-ligand (RANKL) stimulation and is highly expressed in mature osteoclasts. The D5 isoform of Ads is not involved in regulating OCG, as its expression is not induced in response to RANKL. Three clonal Ads knockdown RAW264.7 (RAW) macrophage cell lines with varying degrees of Ads expression and OCG deficiency were generated. The most drastic OCG defect was noted in the clonal cell line with the greatest degree of Ads knockdown as indicated by a lack of TRAcP staining and multinucleation. RNAi mediated knockdown of Ads in osteoclast precursors resulted in distinct morphological changes characterized by altered F-actin distribution and increased filopodia formation. Ads knockdown precursor cells experienced enhanced migration while fusion of knockdown precursors cells was limited. Transient reintroduction of de novo Ads back into the knockdown system was capable of rescuing TRAcP expression but not osteoclast multinucleation most likely due to the transient nature of Ads expression. This preliminary study allows us to conclude that Ads is a RANKL induced early regulator of OCG with a potential role in pre-osteoclast differentiation and fusion. PMID:25275604

  4. The Actin Binding Protein Adseverin Regulates Osteoclastogenesis

    PubMed Central

    Wang, Yongqiang; Kuiper, Johannes W. P.; Glogauer, Michael

    2014-01-01

    Adseverin (Ads), a member of the Gelsolin superfamily of actin binding proteins, regulates the actin cytoskeleton architecture by severing and capping existing filamentous actin (F-actin) strands and nucleating the assembly of new F-actin filaments. Ads has been implicated in cellular secretion, exocytosis and has also been shown to regulate chondrogenesis and megakaryoblastic leukemia cell differentiation. Here we report for the first time that Ads is involved in regulating osteoclastogenesis (OCG). Ads is induced during OCG downstream of RANK-ligand (RANKL) stimulation and is highly expressed in mature osteoclasts. The D5 isoform of Ads is not involved in regulating OCG, as its expression is not induced in response to RANKL. Three clonal Ads knockdown RAW264.7 (RAW) macrophage cell lines with varying degrees of Ads expression and OCG deficiency were generated. The most drastic OCG defect was noted in the clonal cell line with the greatest degree of Ads knockdown as indicated by a lack of TRAcP staining and multinucleation. RNAi mediated knockdown of Ads in osteoclast precursors resulted in distinct morphological changes characterized by altered F-actin distribution and increased filopodia formation. Ads knockdown precursor cells experienced enhanced migration while fusion of knockdown precursors cells was limited. Transient reintroduction of de novo Ads back into the knockdown system was capable of rescuing TRAcP expression but not osteoclast multinucleation most likely due to the transient nature of Ads expression. This preliminary study allows us to conclude that Ads is a RANKL induced early regulator of OCG with a potential role in pre-osteoclast differentiation and fusion. PMID:25275604

  5. The ADF/cofilin family: actin-remodeling proteins

    PubMed Central

    Maciver, Sutherland K; Hussey, Patrick J

    2002-01-01

    The ADF/cofilins are a family of actin-binding proteins expressed in all eukaryotic cells so far examined. Members of this family remodel the actin cytoskeleton, for example during cytokinesis, when the actin-rich contractile ring shrinks as it contracts through the interaction of ADF/cofilins with both monomeric and filamentous actin. The depolymerizing activity is twofold: ADF/cofilins sever actin filaments and also increase the rate at which monomers leave the filament's pointed end. The three-dimensional structure of ADF/cofilins is similar to a fold in members of the gelsolin family of actin-binding proteins in which this fold is typically repeated three or six times; although both families bind polyphosphoinositide lipids and actin in a pH-dependent manner, they share no obvious sequence similarity. Plants and animals have multiple ADF/cofilin genes, belonging in vertebrates to two types, ADF and cofilins. Other eukaryotes (such as yeast, Acanthamoeba and slime moulds) have a single ADF/cofilin gene. Phylogenetic analysis of the ADF/cofilins reveals that, with few exceptions, their relationships reflect conventional views of the relationships between the major groups of organisms. PMID:12049672

  6. Identification of Actin-Binding Proteins from Maize Pollen

    SciTech Connect

    Staiger, C.J.

    2004-01-13

    Specific Aims--The goal of this project was to gain an understanding of how actin filament organization and dynamics are controlled in flowering plants. Specifically, we proposed to identify unique proteins with novel functions by investigating biochemical strategies for the isolation and characterization of actin-binding proteins (ABPs). In particular, our hunt was designed to identify capping proteins and nucleation factors. The specific aims included: (1) to use F-actin affinity chromatography (FAAC) as a general strategy to isolate pollen ABPs (2) to produce polyclonal antisera and perform subcellular localization in pollen tubes (3) to isolate cDNA clones for the most promising ABPs (4) to further purify and characterize ABP interactions with actin in vitro. Summary of Progress By employing affinity chromatography on F-actin or DNase I columns, we have identified at least two novel ABPs from pollen, PrABP80 (gelsolin-like) and ZmABP30, We have also cloned and expressed recombinant protein, as well as generated polyclonal antisera, for 6 interesting ABPs from Arabidopsis (fimbrin AtFIM1, capping protein a/b (AtCP), adenylyl cyclase-associated protein (AtCAP), AtCapG & AtVLN1). We performed quantitative analyses of the biochemical properties for two of these previously uncharacterized ABPs (fimbrin and capping protein). Our studies provide the first evidence for fimbrin activity in plants, demonstrate the existence of barbed-end capping factors and a gelsolin-like severing activity, and provide the quantitative data necessary to establish and test models of F-actin organization and dynamics in plant cells.

  7. Technical advance: identification of plant actin-binding proteins by F-actin affinity chromatography

    NASA Technical Reports Server (NTRS)

    Hu, S.; Brady, S. R.; Kovar, D. R.; Staiger, C. J.; Clark, G. B.; Roux, S. J.; Muday, G. K.

    2000-01-01

    Proteins that interact with the actin cytoskeleton often modulate the dynamics or organization of the cytoskeleton or use the cytoskeleton to control their localization. In plants, very few actin-binding proteins have been identified and most are thought to modulate cytoskeleton function. To identify actin-binding proteins that are unique to plants, the development of new biochemical procedures will be critical. Affinity columns using actin monomers (globular actin, G-actin) or actin filaments (filamentous actin, F-actin) have been used to identify actin-binding proteins from a wide variety of organisms. Monomeric actin from zucchini (Cucurbita pepo L.) hypocotyl tissue was purified to electrophoretic homogeneity and shown to be native and competent for polymerization to actin filaments. G-actin, F-actin and bovine serum albumin affinity columns were prepared and used to separate samples enriched in either soluble or membrane-associated actin-binding proteins. Extracts of soluble actin-binding proteins yield distinct patterns when eluted from the G-actin and F-actin columns, respectively, leading to the identification of a putative F-actin-binding protein of approximately 40 kDa. When plasma membrane-associated proteins were applied to these columns, two abundant polypeptides eluted selectively from the F-actin column and cross-reacted with antiserum against pea annexins. Additionally, a protein that binds auxin transport inhibitors, the naphthylphthalamic acid binding protein, which has been previously suggested to associate with the actin cytoskeleton, was eluted in a single peak from the F-actin column. These experiments provide a new approach that may help to identify novel actin-binding proteins from plants.

  8. Actin-binding proteins: the long road to understanding the dynamic landscape of cellular actin networks.

    PubMed

    Lappalainen, Pekka

    2016-08-15

    The actin cytoskeleton supports a vast number of cellular processes in nonmuscle cells. It is well established that the organization and dynamics of the actin cytoskeleton are controlled by a large array of actin-binding proteins. However, it was only 40 years ago that the first nonmuscle actin-binding protein, filamin, was identified and characterized. Filamin was shown to bind and cross-link actin filaments into higher-order structures and contribute to phagocytosis in macrophages. Subsequently many other nonmuscle actin-binding proteins were identified and characterized. These proteins regulate almost all steps of the actin filament assembly and disassembly cycles, as well as the arrangement of actin filaments into diverse three-dimensional structures. Although the individual biochemical activities of most actin-regulatory proteins are relatively well understood, knowledge of how these proteins function together in a common cytoplasm to control actin dynamics and architecture is only beginning to emerge. Furthermore, understanding how signaling pathways and mechanical cues control the activities of various actin-binding proteins in different cellular, developmental, and pathological processes will keep researchers busy for decades. PMID:27528696

  9. Coactosin-like protein, a human F-actin-binding protein: critical role of lysine-75.

    PubMed Central

    Provost, P; Doucet, J; Stock, A; Gerisch, G; Samuelsson, B; Rådmark, O

    2001-01-01

    Coactosin-like protein (CLP) was recently identified in a yeast two-hybrid screen using 5-lipoxygenase as bait. In the present study, we report the functional characterization of CLP as a human filamentous actin (F-actin)-binding protein. CLP mRNA shows a wide tissue distribution and is predominantly expressed in placenta, lung, kidney and peripheral-blood leucocytes. Endogenous CLP is localized in the cytosol of myeloid cells. Using a two-hybrid approach, actin was identified as a CLP-interacting protein. Binding experiments indicated that CLP associates with F-actin, but does not form a stable complex with globular actin. In transfected mammalian cells, CLP co-localized with actin stress fibres. CLP bound to actin filaments with a stoichiometry of 1:2 (CLP: actin subunits), but could be cross-linked to only one subunit of actin. Site-directed mutagenesis revealed the involvement of Lys(75) of CLP in actin binding, a residue highly conserved in related proteins and supposed to be exposed on the surface of the CLP protein. Our results identify CLP as a new human protein that binds F-actin in vitro and in vivo, and indicate that Lys(75) is essential for this interaction. PMID:11583571

  10. Moesin, ezrin, and p205 are actin-binding proteins associated with neutrophil plasma membranes.

    PubMed Central

    Pestonjamasp, K; Amieva, M R; Strassel, C P; Nauseef, W M; Furthmayr, H; Luna, E J

    1995-01-01

    Actin-binding proteins in bovine neutrophil plasma membranes were identified using blot overlays with 125I-labeled F-actin. Along with surface-biotinylated proteins, membranes were enriched in major actin-binding polypeptides of 78, 81, and 205 kDa. Binding was specific for F-actin because G-actin did not bind. Further, unlabeled F-actin blocked the binding of 125I-labeled F-actin whereas other acidic biopolymers were relatively ineffective. Binding also was specifically inhibited by myosin subfragment 1, but not by CapZ or plasma gelsolin, suggesting that the membrane proteins, like myosin, bind along the sides of the actin filaments. The 78- and 81-kDa polypeptides were identified as moesin and ezrin, respectively, by co-migration on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoprecipitation with antibodies specific for moesin and ezrin. Although not present in detectable amounts in bovine neutrophils, radixin (a third and closely related member of this gene family) also bound 125I-labeled F-actin on blot overlays. Experiments with full-length and truncated bacterial fusion proteins localized the actin-binding site in moesin to the extreme carboxy terminus, a highly conserved sequence. Immunofluorescence micrographs of permeabilized cells and cell "footprints" showed moesin co-localization with actin at the cytoplasmic surface of the plasma membrane, consistent with a role as a membrane-actin-linking protein. Images PMID:7612961

  11. Cloning and sequencing of a gene coding for an actin binding protein of Saccharomyces exiguus.

    PubMed

    Lange, U; Steiner, S; Grolig, F; Wagner, G; Philippsen, P

    1994-03-01

    The actin binding protein Abp1p of the yeast Saccharomyces cervisiae is thought to be involved in the spatial organisation of cell surface growth. It contains a potential actin binding domain and an SH-3 region, a common motif of many signal transduction proteins [1]. We have cloned and sequenced an ABP1 homologous gene of Saccharomyces exiguus, a yeast which is only distantly related to S. cerevisiae. The protein encoded by this gene is slightly larger than the respective S. cerevisiae protein (617 versus 592 amino acids). The two genes are 67.4% identical and the deduced amino acid sequences share an overall identity of 59.8%. The most conserved regions are the 148 N-terminal amino acids containing the potential actin binding site and the 58 C-terminal amino acids including the SH3 domain. In addition, both proteins contain a repeated motif of unknown function which is rich in glutamic acids with the sequence EEEEEEEAPAPSLPSR in the S. exiguus Abp1p. PMID:8110838

  12. Actin-binding proteins coronin-1a and IBA-1 are effective microglial markers for immunohistochemistry.

    PubMed

    Ahmed, Zeshan; Shaw, Gerry; Sharma, Ved P; Yang, Cui; McGowan, Eileen; Dickson, Dennis W

    2007-07-01

    This study identifies the actin-binding protein, coronin-1a, as a novel and effective immunohistochemical marker for microglia in both cell cultures and in formaldehyde-fixed, paraffin-embedded tissue. Antibodies to coronin-1a effectively immunostained microglia in human, monkey, horse, rat, and mouse tissues, even in tissues stored for long periods of time. The identity of coronin-1a-immunoreactive cells as microglia was confirmed using double immunolabeling with cell type-specific markers as well as by morphological features and the distribution of immunoreactive cells. These properties are shared by another actin-binding protein, IBA-1. Unlike IBA-1, coronin-1a immunoreactivity was also detected in lymphocytes and certain other hematopoietic cells. The results indicate that both coronin-1a and IBA-1 are robust markers for microglia that can be used in routinely processed tissue of humans and animals. Because both coronin-1a and IBA-1 are actin-binding proteins that play a role in rearrangement of the membrane cytoskeleton, it suggests that these proteins are critical to dynamic properties of microglia. PMID:17341475

  13. Demonstration in vivo of the role of Arabidopsis PLIM2 actin-binding proteins during pollination.

    PubMed

    Sudo, Keisuke; Park, Jong-In; Sakazono, Satomi; Masuko-Suzuki, Hiromi; Osaka, Masaaki; Kawagishi, Mizuho; Fujita, Kotomi; Maruoka, Mayumi; Nanjo, Hikaru; Suzuki, Go; Suwabe, Keita; Watanabe, Masao

    2013-01-01

    In plant reproduction, pollination is the initial key process in bringing together the male and female gametophytes. When a pollen grain lands on the surface of the stigma, information is exchanged between the pollen and stigmatic cell to determine whether the pollen grain will be accepted or rejected. If it is accepted, the stigmatic papilla cell supplies water and other resources to the pollen for germination and pollen tube elongation. Cellular processes involving actin are essential for pollen germination and tube growth, and actin-binding proteins regulate these processes by interacting with actin filaments to assemble cytoskeletal structures and actin networks. LIM proteins, which belong to a subfamily of cysteine-rich proteins, are a family of actin-binding proteins in plants, and are considered to be important for formation of the actin cytoskeleton and maintenance of its dynamics. Although the physiological and biochemical characteristics of LIMs have been elucidated in vitro in a variety of cell types, their exact role in pollen germination and pollen tube growth during pollination remained unclear. In this manuscript, we focus on the pollen-specific LIM proteins, AtPLIM2a and AtPLIM2c, and define their biological function during pollination in Arabidopsis thaliana. The atplim2a/atplim2c double knockdown RNAi plants showed a reduced pollen germination, approximately one-fifth of wild type, and slower pollen tube growth in the pistil, that is 80.4 μm/hr compared to 140.8 μm/hr in wild type. These defects led to an occasional unfertilized ovule at the bottom of the silique in RNAi plants. Our data provide direct evidence of the biological function of LIM proteins during pollination as actin-binding proteins, modulating cytoskeletal structures and actin networks, and their consequent importance in seed production. PMID:24694391

  14. In vivo dynamics of the F-actin-binding protein neurabin-II.

    PubMed Central

    Stephens, D J; Banting, G

    2000-01-01

    Neurabin-II (spinophilin) is a ubiquitously expressed F-actin-binding protein containing an N-terminal actin-binding domain, a PDZ (PSD95/discs large/ZO-1) domain and a C-terminal domain predicted to form a coiled-coil structure. We have stably expressed a green fluorescent protein (GFP)-tagged version of neurabin-II in PC12 cells, and characterized the in vivo dynamics of this actin-binding protein using confocal fluorescence microscopy. We show that GFP-neurabin-II localizes to actin filaments, especially at cortical sites and areas underlying sites of active membrane remodelling. GFP-neurabin-II labels only a subset of F-actin within these cells, as indicated by rhodamine-phalloidin staining. Both actin filaments and small, highly motile structures within the cell body are seen. Photobleaching experiments show that GFP-neurabin-II also exhibits highly dynamic behaviour when bound to actin filaments. Latrunculin B treatment results in rapid relocalization of GFP-neurabin-II to the cytosol, whereas cytochalasin D treatment causes the collapse of GFP-neurabin-II fluorescence to intensely fluorescent foci of F-actin within the cell body. This collapse is reversed on cytochalasin D removal, recovery from which is greatly accelerated by stimulation of cells with epidermal growth factor (EGF). Furthermore, we show that this EGF-induced relocalization of GFP-neurabin-II is dependent on the activity of the small GTPase Rac1 but not the activity of ADP-ribosylation factor 6. PMID:10620493

  15. Study of the influence of actin-binding proteins using linear analyses of cell deformability.

    PubMed

    Plaza, Gustavo R; Uyeda, Taro Q P; Mirzaei, Zahra; Simmons, Craig A

    2015-07-21

    The actin cytoskeleton plays a key role in the deformability of the cell and in mechanosensing. Here we analyze the contributions of three major actin cross-linking proteins, myosin II, α-actinin and filamin, to cell deformability, by using micropipette aspiration of Dictyostelium cells. We examine the applicability of three simple mechanical models: for small deformation, linear viscoelasticity and drop of liquid with a tense cortex; and for large deformation, a Newtonian viscous fluid. For these models, we have derived linearized equations and we provide a novel, straightforward methodology to analyze the experiments. This methodology allowed us to differentiate the effects of the cross-linking proteins in the different regimes of deformation. Our results confirm some previous observations and suggest important relations between the molecular characteristics of the actin-binding proteins and the cell behavior: the effect of myosin is explained in terms of the relation between the lifetime of the bond to actin and the resistive force; the presence of α-actinin obstructs the deformation of the cytoskeleton, presumably mainly due to the higher molecular stiffness and to the lower dissociation rate constants; and filamin contributes critically to the global connectivity of the network, possibly by rapidly turning over cross-links during the remodeling of the cytoskeletal network, thanks to the higher rate constants, flexibility and larger size. The results suggest a sophisticated relationship between the expression levels of actin-binding proteins, deformability and mechanosensing. PMID:26059185

  16. Regulation of blood-testis barrier by actin binding proteins and protein kinases.

    PubMed

    Li, Nan; Tang, Elizabeth I; Cheng, C Yan

    2016-03-01

    The blood-testis barrier (BTB) is an important ultrastructure in the testis, since the onset of meiosis and spermiogenesis coincides with the establishment of a functional barrier in rodents and humans. It is also noted that a delay in the assembly of a functional BTB following treatment of neonatal rats with drugs such as diethylstilbestrol or adjudin also delays the first wave of spermiation. While the BTB is one of the tightest blood-tissue barriers, it undergoes extensive remodeling, in particular, at stage VIII of the epithelial cycle to facilitate the transport of preleptotene spermatocytes connected in clones across the immunological barrier. Without this timely transport of preleptotene spermatocytes derived from type B spermatogonia, meiosis will be arrested, causing aspermatogenesis. Yet the biology and regulation of the BTB remains largely unexplored since the morphological studies in the 1970s. Recent studies, however, have shed new light on the biology of the BTB. Herein, we critically evaluate some of these findings, illustrating that the Sertoli cell BTB is regulated by actin-binding proteins (ABPs), likely supported by non-receptor protein kinases, to modulate the organization of actin microfilament bundles at the site. Furthermore, microtubule-based cytoskeleton is also working in concert with the actin-based cytoskeleton to confer BTB dynamics. This timely review provides an update on the unique biology and regulation of the BTB based on the latest findings in the field, focusing on the role of ABPs and non-receptor protein kinases. PMID:26628556

  17. Regulation of blood-testis barrier by actin binding proteins and protein kinases

    PubMed Central

    Li, Nan; Tang, Elizabeth I.; Cheng, C. Yan

    2016-01-01

    The blood-testis barrier (BTB) is an important ultrastructure in the testis since the onset of spermatogenesis coincides with the establishment of a functional barrier in rodents and humans. It is also noted that a delay in the assembly of a functional BTB following treatment of neonatal rats with drugs such as diethylstilbestrol or adjudin also delays the first wave of spermiation. While the BTB is one of the tightest blood-tissue barriers, it undergoes extensive remodeling, in particular at stage VIII of the epithelial cycle to facilitate the transport of preleptotene spermatocytes connected in clones across the immunological barrier. Without this timely transport of preleptotene spermatocytes derived from type B spermatogonia, meiosis will be arrested, causing aspermatogenesis. Yet the biology and regulation of the BTB remains largely unexplored since the morphological studies in the 1970s. Recent studies, however, have shed new light on the biology of the BTB. Herein, we critically evaluate some of these findings, illustrating that the Sertoli cell BTB is regulated by actin binding proteins (ABPs), likely supported by non-receptor protein kinases, to modulate the organization of actin microfilament bundles at the site. Furthermore, microtubule (MT)-based cytoskeleton is also working in concert with the actin-based cytoskeleton to confer BTB dynamics. This timely review provides an update on the unique biology and regulation of the BTB based on the latest findings in the field, focusing on the role of ABPs and non-receptor protein kinases. PMID:26628556

  18. Functional characterization of spectrin-actin-binding domains in 4.1 family of proteins.

    PubMed

    Gimm, J Aura; An, Xiuli; Nunomura, Wataru; Mohandas, Narla

    2002-06-11

    Protein 4.1R is the prototypical member of a protein family that includes 4.1G, 4.1B, and 4.1N. 4.1R plays a crucial role in maintaining membrane mechanical integrity by binding cooperatively to spectrin and actin through its spectrin-actin-binding (SAB) domain. While the binary interaction between 4.1R and spectrin has been well characterized, the actin binding site in 4.1R remains unidentified. Moreover, little is known about the interaction of 4.1R homologues with spectrin and actin. In the present study, we showed that the 8 aa motif (LKKNFMES) within the 10 kDa spectrin-actin-binding domain of 4.1R plays a critical role in binding of 4.1R to actin. Recombinant 4.1R SAB domain peptides with mutations in this motif showed a marked decrease in their ability to form ternary complexes with spectrin and actin. Binary protein-protein interaction studies revealed that this decrease resulted from the inability of mutant SAB peptides to bind to actin filaments while affinity for spectrin was unchanged. We also documented that the 14 C-terminal residues of the 21 amino acid cassette encoded by exon 16 in conjunction with residues 27-43 encoded by exon 17 constituted a fully functional minimal spectrin-binding motif. Finally, we showed that 4.1N SAB domain was unable to form a ternary complex with spectrin and actin, while 4.1G and 4.1B SAB domains were able to form such a complex but less efficiently than 4.1R SAB. This was due to a decrease in the ability of 4.1G and 4.1B SAB domain to interact with actin but not with spectrin. These data enabled us to propose a model for the 4.1R-spectrin-actin ternary complex which may serve as a general paradigm for regulation of spectrin-based cytoskeleton interaction in various cell types. PMID:12044158

  19. The Actin-Binding Protein α-Adducin Is Required for Maintaining Axon Diameter.

    PubMed

    Leite, Sérgio Carvalho; Sampaio, Paula; Sousa, Vera Filipe; Nogueira-Rodrigues, Joana; Pinto-Costa, Rita; Peters, Luanne Laurel; Brites, Pedro; Sousa, Mónica Mendes

    2016-04-19

    The actin-binding protein adducin was recently identified as a component of the neuronal subcortical cytoskeleton. Here, we analyzed mice lacking adducin to uncover the function of this protein in actin rings. α-adducin knockout mice presented progressive axon enlargement in the spinal cord and optic and sciatic nerves, followed by axon degeneration and loss. Using stimulated emission depletion super-resolution microscopy, we show that a periodic subcortical actin cytoskeleton is assembled in every neuron type inspected including retinal ganglion cells and dorsal root ganglia neurons. In neurons devoid of adducin, the actin ring diameter increased, although the inter-ring periodicity was maintained. In vitro, the actin ring diameter adjusted as axons grew, suggesting the lattice is dynamic. Our data support a model in which adducin activity is not essential for actin ring assembly and periodicity but is necessary to control the diameter of both actin rings and axons and actin filament growth within rings. PMID:27068466

  20. Rho, nuclear actin, and actin-binding proteins in the regulation of transcription and gene expression

    PubMed Central

    Rajakylä, Eeva Kaisa; Vartiainen, Maria K

    2014-01-01

    Actin cytoskeleton is one of the main targets of Rho GTPases, which act as molecular switches on many signaling pathways. During the past decade, actin has emerged as an important regulator of gene expression. Nuclear actin plays a key role in transcription, chromatin remodeling, and pre-mRNA processing. In addition, the “status” of the actin cytoskeleton is used as a signaling intermediate by at least the MKL1-SRF and Hippo-pathways, which culminate in the transcriptional regulation of cytoskeletal and growth-promoting genes, respectively. Rho GTPases may therefore regulate gene expression by controlling either cytoplasmic or nuclear actin dynamics. Although the regulation of nuclear actin polymerization is still poorly understood, many actin-binding proteins, which are downstream effectors of Rho, are found in the nuclear compartment. In this review, we discuss the possible mechanisms and key proteins that may mediate the transcriptional regulation by Rho GTPases through actin. PMID:24603113

  1. Rho, nuclear actin, and actin-binding proteins in the regulation of transcription and gene expression.

    PubMed

    Rajakylä, Eeva Kaisa; Vartiainen, Maria K

    2014-01-01

    Actin cytoskeleton is one of the main targets of Rho GTPases, which act as molecular switches on many signaling pathways. During the past decade, actin has emerged as an important regulator of gene expression. Nuclear actin plays a key role in transcription, chromatin remodeling, and pre-mRNA processing. In addition, the "status" of the actin cytoskeleton is used as a signaling intermediate by at least the MKL1-SRF and Hippo-pathways, which culminate in the transcriptional regulation of cytoskeletal and growth-promoting genes, respectively. Rho GTPases may therefore regulate gene expression by controlling either cytoplasmic or nuclear actin dynamics. Although the regulation of nuclear actin polymerization is still poorly understood, many actin-binding proteins, which are downstream effectors of Rho, are found in the nuclear compartment. In this review, we discuss the possible mechanisms and key proteins that may mediate the transcriptional regulation by Rho GTPases through actin. PMID:24603113

  2. Regulation of water flow by actin-binding protein-induced actin gelatin.

    PubMed Central

    Ito, T; Suzuki, A; Stossel, T P

    1992-01-01

    Actin filaments inhibit osmotically driven water flow (Ito, T., K.S. Zaner, and T.P. Stossel. 1987. Biophys. J. 51: 745-753). Here we show that the actin gelation protein, actin-binding protein (ABP), impedes both osmotic shrinkage and swelling of an actin filament solution and reduces markedly the concentration of actin filaments required for this inhibition. These effects depend on actin filament immobilization, because the ABP concentration that causes initial impairment of water flow by actin filaments corresponds to the gel point measured viscometrically and because gelsolin, which noncovalently severs actin filaments, solates actin gels and restores water flow in a solution of actin cross-linked by ABP. Since ABP gels actin filaments in the periphery of many eukaryotic cells, such actin networks may contribute to physiological cell volume regulation. PMID:1318095

  3. Human endothelial actin-binding protein (ABP-280, nonmuscle filamin): a molecular leaf spring

    PubMed Central

    1990-01-01

    Actin-binding protein (ABP-280, nonmuscle filamin) is a ubiquitous dimeric actin cross-linking phosphoprotein of peripheral cytoplasm, where it promotes orthogonal branching of actin filaments and links actin filaments to membrane glycoproteins. The complete nucleotide sequence of human endothelial cell ABP cDNA predicts a polypeptide subunit chain of 2,647 amino acids, corresponding to 280 kD, also the mass derived from physical measurements of the native protein. The actin-binding domain is near the amino-terminus of the subunit where the amino acid sequence is similar to other actin filament binding proteins, including alpha-actinin, beta-spectrin, dystrophin, and Dictyostelium abp-120. The remaining 90% of the sequence comprises 24 repeats, each approximately 96 residues long, predicted to have stretches of beta-sheet secondary structure interspersed with turns. The first 15 repeats may have substantial intrachain hydrophobic interactions and overlap in a staggered fashion to yield a backbone with mechanical resilience. Sequence insertions immediately before repeats 16 and 24 predict two hinges in the molecule near points where rotary-shadowed molecules appear to swivel in electron micrographs. Both putative hinge regions are susceptible to cleavage by proteases and the second also contains the site that binds the platelet glycoprotein Ib/IX complex. Phosphorylation consensus sequences are also located in the hinges or near them. Degeneracy within every even- numbered repeat between 16 and 24 and the insertion before repeat 24 may convert interactions within chains to interactions between chains to account for dimer formation within a domain of 7 kD at the carboxy- terminus. The structure of ABP dimers resembles a leaf spring. Interchain interactions hold the leaves firmly together at one end, whereas intrachain hydrophobic bonds reinforce the arms of the spring where the leaves diverge, making it sufficiently stiff to promote high- angle branching of actin

  4. Human endothelial actin-binding protein (ABP-280, nonmuscle filamin): a molecular leaf spring.

    PubMed

    Gorlin, J B; Yamin, R; Egan, S; Stewart, M; Stossel, T P; Kwiatkowski, D J; Hartwig, J H

    1990-09-01

    Actin-binding protein (ABP-280, nonmuscle filamin) is a ubiquitous dimeric actin cross-linking phosphoprotein of peripheral cytoplasm, where it promotes orthogonal branching of actin filaments and links actin filaments to membrane glycoproteins. The complete nucleotide sequence of human endothelial cell ABP cDNA predicts a polypeptide subunit chain of 2,647 amino acids, corresponding to 280 kD, also the mass derived from physical measurements of the native protein. The actin-binding domain is near the amino-terminus of the subunit where the amino acid sequence is similar to other actin filament binding proteins, including alpha-actinin, beta-spectrin, dystrophin, and Dictyostelium abp-120. The remaining 90% of the sequence comprises 24 repeats, each approximately 96 residues long, predicted to have stretches of beta-sheet secondary structure interspersed with turns. The first 15 repeats may have substantial intrachain hydrophobic interactions and overlap in a staggered fashion to yield a backbone with mechanical resilience. Sequence insertions immediately before repeats 16 and 24 predict two hinges in the molecule near points where rotary-shadowed molecules appear to swivel in electron micrographs. Both putative hinge regions are susceptible to cleavage by proteases and the second also contains the site that binds the platelet glycoprotein Ib/IX complex. Phosphorylation consensus sequences are also located in the hinges or near them. Degeneracy within every even-numbered repeat between 16 and 24 and the insertion before repeat 24 may convert interactions within chains to interactions between chains to account for dimer formation within a domain of 7 kD at the carboxy-terminus. The structure of ABP dimers resembles a leaf spring. Interchain interactions hold the leaves firmly together at one end, whereas intrachain hydrophobic bonds reinforce the arms of the spring where the leaves diverge, making it sufficiently stiff to promote high-angle branching of actin

  5. Dephosphorylation of cofilin in stimulated platelets: roles for a GTP-binding protein and Ca2+.

    PubMed Central

    Davidson, M M; Haslam, R J

    1994-01-01

    In human platelets, thrombin not only stimulates the phosphorylation of pleckstrin (P47) and of myosin P-light chains, but also induces the dephosphorylation of an 18-19 kDa phosphoprotein (P18) [Imaoka, Lynham and Haslam (1983) J. Biol. Chem. 258, 11404-11414]. We have now studied this protein in detail. The thrombin-induced dephosphorylation reaction did not begin until the phosphorylation of myosin P-light chains and the secretion of dense-granule 5-hydroxytryptamine were nearly complete, but did parallel the later stages of platelet aggregation. Experiments with ionophore A23187 and phorbol 12-myristate 13-acetate indicated that dephosphorylation of P18 was stimulated by Ca2+, but not by protein kinase C. Two-dimensional analysis of platelet proteins, using non-equilibrium pH gradient electrophoresis followed by SDS/PAGE, showed that thrombin decreased the amount of phosphorylated P18 in platelets by up to 70% and slightly increased the amount of a more basic unlabelled protein that was present in 3-fold excess of P18 in unstimulated platelets. These two proteins were identified as the phosphorylated and non-phosphorylated forms of the pH-sensitive actin-depolymerizing protein, cofilin, by sequencing of peptide fragments and immunoblotting with a monoclonal antibody specific for cofilin. The molar concentration of cofilin in platelets was approx. 10% that of actin. Platelet cofilin was phosphorylated exclusively on serine. Experiments with electropermeabilized platelets showed that dephosphorylation of cofilin could be stimulated by guanosine 5'-[gamma-thio]triphosphate (GTP[S]) in the absence of Ca2+ or by a free Ca2+ concentration of 10 microM. This GTP[S]-induced dephosphorylation reaction was inhibited by 1-naphthyl phosphate, but not by okadaic acid. Our results add cofilin to the actin-binding proteins that may regulate the platelet cytoskeleton, and suggest that platelet cofilin can be activated by dephosphorylation reactions initiated either by a GTP

  6. Modification of Cys-837 identifies an actin-binding site in the beta-propeller protein scruin.

    PubMed Central

    Sun, S; Footer, M; Matsudaira, P

    1997-01-01

    In the acrosomal process of Limulus sperm, the beta-propeller protein scruin cross-links actin into a crystalline bundle. To confirm that scruin has the topology of a beta-propeller protein and to understand how scruin binds actin, we compared the solvent accessibility of cysteine residues in scruin and the acrosomal process by chemical modification with (1,5-IAEDANS). In soluble scruin, the two most reactive cysteines of soluble scruin are C837 and C900, whereas C146, C333, and C683 are moderately reactive. This pattern of reactivity is consistent with the topology of a typical beta-propeller protein; all of the reactive cysteines map to putative loops and turns whereas the unreactive cysteines lie within the predicted interior of the protein. The chemical reactivities of cysteine in the acrosomal process implicate C837 at an actin-binding site. In contrast to soluble scruin, in the acrosomal process, C837 is completely unreactive while the other cysteines become less reactive. Binding studies of chemically modified scruin correlate the extent of modification at C837 with the extent of inhibition of actin binding. Furthermore, peptides corresponding to residues flanking C837 bind actin and narrow a possible actin-binding region to a KQK sequence. On the basis of these studies, our results suggest that an actin-binding site lies in the C-terminal domain of scruin and involves a putative loop defined by C837. Images PMID:9188095

  7. Cofilin-induced cooperative conformational changes of actin subunits revealed using cofilin-actin fusion protein

    PubMed Central

    Umeki, Nobuhisa; Hirose, Keiko; Uyeda, Taro Q. P.

    2016-01-01

    To investigate cooperative conformational changes of actin filaments induced by cofilin binding, we engineered a fusion protein made of Dictyostelium cofilin and actin. The filaments of the fusion protein were functionally similar to actin filaments bound with cofilin in that they did not bind rhodamine-phalloidin, had quenched fluorescence of pyrene attached to Cys374 and showed enhanced susceptibility of the DNase loop to cleavage by subtilisin. Quantitative analyses of copolymers made of different ratios of the fusion protein and control actin further demonstrated that the fusion protein affects the structure of multiple neighboring actin subunits in copolymers. Based on these and other recent related studies, we propose a mechanism by which conformational changes induced by cofilin binding is propagated unidirectionally to the pointed ends of the filaments, and cofilin clusters grow unidirectionally to the pointed ends following this path. Interestingly, the fusion protein was unable to copolymerize with control actin at pH 6.5 and low ionic strength, suggesting that the structural difference between the actin moiety in the fusion protein and control actin is pH-sensitive. PMID:26842224

  8. The actin-binding ERM protein Moesin directly regulates spindle assembly and function during mitosis.

    PubMed

    Vilmos, Péter; Kristó, Ildikó; Szikora, Szilárd; Jankovics, Ferenc; Lukácsovich, Tamás; Kari, Beáta; Erdélyi, Miklós

    2016-06-01

    Ezrin-Radixin-Moesin proteins are highly conserved, actin-binding cytoskeletal proteins that play an essential role in microvilli formation, T-cell activation, and tumor metastasis by linking actin filaments to the plasma membrane. Recent studies demonstrated that the only Ezrin-Radixin-Moesin protein of Drosophila melanogaster, Moesin, is involved in mitotic spindle function through stabilizing cell shape and microtubules at the cell cortex. We previously observed that Moesin localizes to the mitotic spindle; hence, we tested for the biological significance of this surprising localization and investigated whether it plays a direct role in spindle function. To separate the cortical and spindle functions of Moesin during mitosis we combined cell biological and genetic methods. We used early Drosophila embryos, in which mitosis occurs in the absence of a cell cortex, and found in vivo evidence for the direct requirement of Moesin in mitotic spindle assembly and function. We also found that the accumulation of Moesin precedes the construction of the microtubule spindle, and the fusiform structure formed by Moesin persists even after the microtubules have disassembled. PMID:27006187

  9. Mutations in the Gene That Encodes the F-Actin Binding Protein Anillin Cause FSGS

    PubMed Central

    Hall, Gentzon; Adeyemo, Adebowale; Hanke, Nils; Tossidou, Irini; Burchette, James; Wu, Guanghong; Homstad, Alison; Sparks, Matthew A.; Gomez, Jose; Jiang, Ruiji; Alonso, Andrea; Lavin, Peter; Conlon, Peter; Korstanje, Ron; Stander, M. Christine; Shamsan, Ghaidan; Barua, Moumita; Spurney, Robert; Singhal, Pravin C.; Kopp, Jeffrey B.; Haller, Hermann; Howell, David; Pollak, Martin R.; Shaw, Andrey S.; Schiffer, Mario; Winn, Michelle P.

    2014-01-01

    FSGS is characterized by segmental scarring of the glomerulus and is a leading cause of kidney failure. Identification of genes causing FSGS has improved our understanding of disease mechanisms and points to defects in the glomerular epithelial cell, the podocyte, as a major factor in disease pathogenesis. Using a combination of genome-wide linkage studies and whole-exome sequencing in a kindred with familial FSGS, we identified a missense mutation R431C in anillin (ANLN), an F-actin binding cell cycle gene, as a cause of FSGS. We screened 250 additional families with FSGS and found another variant, G618C, that segregates with disease in a second family with FSGS. We demonstrate upregulation of anillin in podocytes in kidney biopsy specimens from individuals with FSGS and kidney samples from a murine model of HIV-1–associated nephropathy. Overexpression of R431C mutant ANLN in immortalized human podocytes results in enhanced podocyte motility. The mutant anillin displays reduced binding to the slit diaphragm–associated scaffold protein CD2AP. Knockdown of the ANLN gene in zebrafish morphants caused a loss of glomerular filtration barrier integrity, podocyte foot process effacement, and an edematous phenotype. Collectively, these findings suggest that anillin is important in maintaining the integrity of the podocyte actin cytoskeleton. PMID:24676636

  10. Wnt Signalling Promotes Actin Dynamics during Axon Remodelling through the Actin-Binding Protein Eps8

    PubMed Central

    Salinas, Patricia C.

    2015-01-01

    Upon arrival at their synaptic targets, axons slow down their growth and extensively remodel before the assembly of presynaptic boutons. Wnt proteins are target-derived secreted factors that promote axonal remodelling and synaptic assembly. In the developing spinal cord, Wnts secreted by motor neurons promote axonal remodelling of NT-3 responsive dorsal root ganglia neurons. Axon remodelling induced by Wnts is characterised by growth cone pausing and enlargement, processes that depend on the re-organisation of microtubules. However, the contribution of the actin cytoskeleton has remained unexplored. Here, we demonstrate that Wnt3a regulates the actin cytoskeleton by rapidly inducing F-actin accumulation in growth cones from rodent DRG neurons through the scaffold protein Dishevelled-1 (Dvl1) and the serine-threonine kinase Gsk3β. Importantly, these changes in actin cytoskeleton occurs before enlargement of the growth cones is evident. Time-lapse imaging shows that Wnt3a increases lamellar protrusion and filopodia velocity. In addition, pharmacological inhibition of actin assembly demonstrates that Wnt3a increases actin dynamics. Through a yeast-two hybrid screen, we identified the actin-binding protein Eps8 as a direct interactor of Dvl1, a scaffold protein crucial for the Wnt signalling pathway. Gain of function of Eps8 mimics Wnt-mediated axon remodelling, whereas Eps8 silencing blocks the axon remodelling activity of Wnt3a. Importantly, blockade of the Dvl1-Eps8 interaction completely abolishes Wnt3a-mediated axonal remodelling. These findings demonstrate a novel role for Wnt-Dvl1 signalling through Eps8 in the regulation of axonal remodeling. PMID:26252776

  11. Actin-binding protein G (AbpG) participates in modulating the actin cytoskeleton and cell migration in Dictyostelium discoideum

    PubMed Central

    Lin, Wei-Chi; Wang, Liang-Chen; Pang, Te-Ling; Chen, Mei-Yu

    2015-01-01

    Cell migration is involved in various physiological and pathogenic events, and the complex underlying molecular mechanisms have not been fully elucidated. The simple eukaryote Dictyostelium discoideum displays chemotactic locomotion in stages of its life cycle. By characterizing a Dictyostelium mutant defective in chemotactic responses, we identified a novel actin-binding protein serving to modulate cell migration and named it actin-binding protein G (AbpG); this 971–amino acid (aa) protein contains an N-terminal type 2 calponin homology (CH2) domain followed by two large coiled-coil regions. In chemoattractant gradients, abpG− cells display normal directional persistence but migrate significantly more slowly than wild-type cells; expressing Flag-AbpG in mutant cells eliminates the motility defect. AbpG is enriched in cortical/lamellipodial regions and colocalizes well with F-actin; aa 401–600 and aa 501–550 fragments of AbpG show the same distribution as full-length AbpG. The aa 501–550 region of AbpG, which is essential for AbpG to localize to lamellipodia and to rescue the phenotype of abpG− cells, is sufficient for binding to F-actin and represents a novel actin-binding protein domain. Compared with wild-type cells, abpG− cells have significantly higher F-actin levels. Collectively our results suggest that AbpG may participate in modulating actin dynamics to optimize cell locomotion. PMID:25609090

  12. Actin-binding protein G (AbpG) participates in modulating the actin cytoskeleton and cell migration in Dictyostelium discoideum.

    PubMed

    Lin, Wei-Chi; Wang, Liang-Chen; Pang, Te-Ling; Chen, Mei-Yu

    2015-03-15

    Cell migration is involved in various physiological and pathogenic events, and the complex underlying molecular mechanisms have not been fully elucidated. The simple eukaryote Dictyostelium discoideum displays chemotactic locomotion in stages of its life cycle. By characterizing a Dictyostelium mutant defective in chemotactic responses, we identified a novel actin-binding protein serving to modulate cell migration and named it actin-binding protein G (AbpG); this 971-amino acid (aa) protein contains an N-terminal type 2 calponin homology (CH2) domain followed by two large coiled-coil regions. In chemoattractant gradients, abpG(-) cells display normal directional persistence but migrate significantly more slowly than wild-type cells; expressing Flag-AbpG in mutant cells eliminates the motility defect. AbpG is enriched in cortical/lamellipodial regions and colocalizes well with F-actin; aa 401-600 and aa 501-550 fragments of AbpG show the same distribution as full-length AbpG. The aa 501-550 region of AbpG, which is essential for AbpG to localize to lamellipodia and to rescue the phenotype of abpG(-) cells, is sufficient for binding to F-actin and represents a novel actin-binding protein domain. Compared with wild-type cells, abpG(-) cells have significantly higher F-actin levels. Collectively our results suggest that AbpG may participate in modulating actin dynamics to optimize cell locomotion. PMID:25609090

  13. An actin cytoskeleton with evolutionarily conserved functions in the absence of canonical actin-binding proteins

    PubMed Central

    Paredez, Alexander R.; Assaf, Zoe June; Sept, David; Timofejeva, Ljudmilla; Dawson, Scott C.; Wang, Chung-Ju Rachel; Cande, W. Z.

    2011-01-01

    Giardia intestinalis, a human intestinal parasite and member of what is perhaps the earliest-diverging eukaryotic lineage, contains the most divergent eukaryotic actin identified to date and is the first eukaryote known to lack all canonical actin-binding proteins (ABPs). We sought to investigate the properties and functions of the actin cytoskeleton in Giardia to determine whether Giardia actin (giActin) has reduced or conserved roles in core cellular processes. In vitro polymerization of giActin produced filaments, indicating that this divergent actin is a true filament-forming actin. We generated an anti-giActin antibody to localize giActin throughout the cell cycle. GiActin localized to the cortex, nuclei, internal axonemes, and formed C-shaped filaments along the anterior of the cell and a flagella-bundling helix. These structures were regulated with the cell cycle and in encysting cells giActin was recruited to the Golgi-like cyst wall processing vesicles. Knockdown of giActin demonstrated that giActin functions in cell morphogenesis, membrane trafficking, and cytokinesis. Additionally, Giardia contains a single G protein, giRac, which affects the Giardia actin cytoskeleton independently of known target ABPs. These results imply that there exist ancestral and perhaps conserved roles for actin in core cellular processes that are independent of canonical ABPs. Of medical significance, the divergent giActin cytoskeleton is essential and commonly used actin-disrupting drugs do not depolymerize giActin structures. Therefore, the giActin cytoskeleton is a promising drug target for treating giardiasis, as we predict drugs that interfere with the Giardia actin cytoskeleton will not affect the mammalian host. PMID:21444821

  14. Polycystin-2 (TRPP2) Regulation by Ca2+ Is Effected and Diversified by Actin-Binding Proteins

    PubMed Central

    Cantero, María del Rocío; Cantiello, Horacio F.

    2015-01-01

    Calcium regulation of Ca2+-permeable ion channels is an important mechanism in the control of cell function. Polycystin-2 (PC2, TRPP2), a member of the transient receptor potential superfamily, is a nonselective cation channel with Ca2+ permeability. The molecular mechanisms associated with PC2 regulation by Ca2+ remain ill-defined. We recently demonstrated that PC2 from human syncytiotrophoblast (PC2hst) but not the in vitro translated protein (PC2iv), functionally responds to changes in intracellular (cis) Ca2+. In this study we determined the regulatory effect(s) of Ca2+-sensitive and -insensitive actin-binding proteins (ABPs) on PC2iv channel function in a lipid bilayer system. The actin-bundling protein α-actinin increased PC2iv channel function in the presence of cis Ca2+, although instead was inhibitory in its absence. Conversely, filamin that shares actin-binding domains with α-actinin had a strong inhibitory effect on PC2iv channel function in the presence, but no effect in the absence of cis Ca2+. Gelsolin stimulated PC2iv channel function in the presence, but not the absence of cis Ca2+. In contrast, profilin that shares actin-binding domains with gelsolin, significantly increased PC2iv channel function both in the presence and absence of Ca2+. The distinct effect(s) of the ABPs on PC2iv channel function demonstrate that Ca2+ regulation of PC2 is actually mediated by direct interaction(s) with structural elements of the actin cytoskeleton. These data indicate that specific ABP-PC2 complexes would confer distinct Ca2+-sensitive properties to the channel providing functional diversity to the cytoskeletal control of transient receptor potential channel regulation. PMID:25954877

  15. MARCKS is a natively unfolded protein with an inaccessible actin-binding site: evidence for long-range intramolecular interactions.

    PubMed

    Tapp, Hazel; Al-Naggar, Iman M; Yarmola, Elena G; Harrison, Alexis; Shaw, Gerry; Edison, Arthur S; Bubb, Michael R

    2005-03-18

    Myristoylated alanine-rich C kinase substrate (MARCKS) is an unfolded protein that contains well characterized actin-binding sites within the phosphorylation site domain (PSD), yet paradoxically, we now find that intact MARCKS does not bind to actin. Intact MARCKS also does not bind as well to calmodulin as does the PSD alone. Myristoylation at the N terminus alters how calmodulin binds to MARCKS, implying that, despite its unfolded state, the distant N terminus influences binding events at the PSD. We show that the free PSD binds with site specificity to MARCKS, suggesting that long-range intramolecular interactions within MARCKS are also possible. Because of the unusual primary sequence of MARCKS with an overall isoelectric point of 4.2 yet a very basic PSD (overall charge of +13), we speculated that ionic interactions between oppositely charged domains of MARCKS were responsible for long-range interactions within MARCKS that sterically influence binding events at the PSD and that explain the observed differences between properties of the PSD and MARCKS. Consistent with this hypothesis, chemical modifications of MARCKS that neutralize negatively charged residues outside of the PSD allow the PSD to bind to actin and increase the affinity of MARCKS for calmodulin. Similarly, both myristoylation of MARCKS and cleavage of MARCKS by calpain are shown to increase the availability of the PSD so as to activate its actin-binding activity. Because abundant evidence supports the conclusion that MARCKS is an important protein in regulating actin dynamics, our data imply that post-translational modifications of MARCKS are necessary and sufficient to regulate actin-binding activity. PMID:15640140

  16. Heterodimeric Capping Protein from Arabidopsis Is a Membrane-Associated, Actin-Binding Protein1[W][OPEN

    PubMed Central

    Jimenez-Lopez, Jose C.; Wang, Xia; Kotchoni, Simeon O.; Huang, Shanjin; Szymanski, Daniel B.; Staiger, Christopher J.

    2014-01-01

    The actin cytoskeleton is a major regulator of cell morphogenesis and responses to biotic and abiotic stimuli. The organization and activities of the cytoskeleton are choreographed by hundreds of accessory proteins. Many actin-binding proteins are thought to be stimulus-response regulators that bind to signaling phospholipids and change their activity upon lipid binding. Whether these proteins associate with and/or are regulated by signaling lipids in plant cells remains poorly understood. Heterodimeric capping protein (CP) is a conserved and ubiquitous regulator of actin dynamics. It binds to the barbed end of filaments with high affinity and modulates filament assembly and disassembly reactions in vitro. Direct interaction of CP with phospholipids, including phosphatidic acid, results in uncapping of filament ends in vitro. Live-cell imaging and reverse-genetic analyses of cp mutants in Arabidopsis (Arabidopsis thaliana) recently provided compelling support for a model in which CP activity is negatively regulated by phosphatidic acid in vivo. Here, we used complementary biochemical, subcellular fractionation, and immunofluorescence microscopy approaches to elucidate CP-membrane association. We found that CP is moderately abundant in Arabidopsis tissues and present in a microsomal membrane fraction. Sucrose density gradient separation and immunoblotting with known compartment markers were used to demonstrate that CP is enriched on membrane-bound organelles such as the endoplasmic reticulum and Golgi. This association could facilitate cross talk between the actin cytoskeleton and a wide spectrum of essential cellular functions such as organelle motility and signal transduction. PMID:25201878

  17. Structure of the ERM protein moesin reveals the FERM domain fold masked by an extended actin binding tail domain.

    PubMed

    Pearson, M A; Reczek, D; Bretscher, A; Karplus, P A

    2000-04-28

    The ezrin-radixin-moesin (ERM) protein family link actin filaments of cell surface structures to the plasma membrane, using a C-terminal F-actin binding segment and an N-terminal FERM domain, a common membrane binding module. ERM proteins are regulated by an intramolecular association of the FERM and C-terminal tail domains that masks their binding sites. The crystal structure of a dormant moesin FERM/tail complex reveals that the FERM domain has three compact lobes including an integrated PTB/PH/ EVH1 fold, with the C-terminal segment bound as an extended peptide masking a large surface of the FERM domain. This extended binding mode suggests a novel mechanism for how different signals could produce varying levels of activation. Sequence conservation suggests a similar regulation of the tumor suppressor merlin. PMID:10847681

  18. Drebrin inhibits cofilin-induced severing of F-actin.

    PubMed

    Grintsevich, Elena E; Reisler, Emil

    2014-08-01

    Molecular cross-talk between neuronal drebrin A and cofilin is believed to be a part of the activity-dependent cytoskeleton-modulating pathway in dendritic spines. Impairments in this pathway are implicated also in synaptic dysfunction in Alzheimer's disease, Down syndrome, epilepsy, and normal aging. However, up to now the molecular interplay between cofilin and drebrin has not been elucidated. TIRF microscopy and solution experiments revealed that full length drebrin A or its actin binding core (Drb1-300) inhibits, but do not abolish cofilin-induced severing of actin filaments. Cosedimentation experiments showed that F-actin can be fully occupied with combination of these two proteins. The dependence of cofilin binding on fractional saturation of actin filaments with drebrin suggests direct competition between these two proteins for F-actin binding. This implies that cofilin and drebrin can either overcome or reverse the allosteric changes in F-actin induced by the competitor's binding. The ability of cofilin to displace drebrin from actin filaments is pH dependent and is facilitated at acidic pH (6.8). Pre-steady state kinetic experiments reveal that both binding and dissociation of drebrin to/from actin filaments is faster than that reported for cooperative binding of cofilin. We found, that drebrin displacement by cofilin is greatly inhibited when actin severing is abolished, which might be linked to the cooperativity of drebrin binding to actin filaments. Our results contribute to molecular understanding of the competitive interactions of drebrin and cofilin with actin filaments. PMID:25047716

  19. [Molecular mechanisms for collective cell migration--perspectives and approaches from the studies on the actin-binding protein Girdin].

    PubMed

    Enomoto, Atsushi; Kato, Takuya; Asai, Naoya; Takahashi, Masahide

    2016-03-01

    In embryonal development and pathogenesis of diseases, cells often get connected and form small groups to undergo "collective migration", rather than spread out individually. The examples include the migration of neural crest cells and neuroblasts during development and the invasion of cancers in surrounding stroma, indicating the importance and significance of collective behavior of cells in the body. Recent studies have revealed the mechanisms for collective cell migration, which had seemed not to be the subject of traditional cell biology on single cells in culture. The heterogeneity in cell groups is also a key in understanding the mechanisms for collective cell migration. In this article, we describe recently emerging mechanisms for collective cell migration, with a particular focus on our studies on the actin-binding protein Girdin and tripartite motif containing 27. PMID:27025099

  20. Functional characterization of protein 4.1 homolog in amphioxus: defining a cryptic spectrin-actin-binding site.

    PubMed

    Wang, Lixia; Wang, Yuan; Li, Zhaohe; Gao, Zhan; Zhang, Shicui

    2013-01-01

    Vertebrate 4.1 proteins have a spectrin-actin-binding (SAB) domain, which is lacking in all the invertebrate 4.1 proteins indentified so far, and it was therefore proposed that the SAB domain emerged with the advent of vertebrates during evolution. Here we demonstrated for the first time that amphioxus (an invertebrate chordate) protein 4.1, though lacking a recognizable SAB, was able to bind both spectrin and actin, with a binding capacity comparable to that of human protein 4.1. Detailed structure-activity analyses revealed that the unique domain U2/3 was a newly identified SAB-like domain capable of interacting with spectrin and actin, suggesting the presence of a "cryptic" SAB domain in amphioxus 4.1 protein. We also showed that amphioxus 4.1 protein gene was the common ancestor of vertebrate 4.1 protein genes, from which 4.1R, 4.1N, 4.1G, and 4.1B genes originated. This work will encourage further study on the structure-activity of invertebrate 4.1 protein and its interacting proteins. PMID:24096627

  1. Functional characterization of protein 4.1 homolog in amphioxus: Defining a cryptic spectrin-actin-binding site

    PubMed Central

    Wang, Lixia; Wang, Yuan; Li, Zhaohe; Gao, Zhan; Zhang, Shicui

    2013-01-01

    Vertebrate 4.1 proteins have a spectrin-actin-binding (SAB) domain, which is lacking in all the invertebrate 4.1 proteins indentified so far, and it was therefore proposed that the SAB domain emerged with the advent of vertebrates during evolution. Here we demonstrated for the first time that amphioxus (an invertebrate chordate) protein 4.1, though lacking a recognizable SAB, was able to bind both spectrin and actin, with a binding capacity comparable to that of human protein 4.1. Detailed structure-activity analyses revealed that the unique domain U2/3 was a newly identified SAB-like domain capable of interacting with spectrin and actin, suggesting the presence of a “cryptic” SAB domain in amphioxus 4.1 protein. We also showed that amphioxus 4.1 protein gene was the common ancestor of vertebrate 4.1 protein genes, from which 4.1R, 4.1N, 4.1G, and 4.1B genes originated. This work will encourage further study on the structure-activity of invertebrate 4.1 protein and its interacting proteins. PMID:24096627

  2. Cooperative and non-cooperative conformational changes of F-actin induced by cofilin

    SciTech Connect

    Aihara, Tomoki; Oda, Toshiro

    2013-05-31

    Highlights: •Mobility of MTSL attached to C374 in F-actin became high upon addition of cofilin. •Change of motility of MTSL attached to C374 with cofilin-binding was cooperative. •Mobility of MTSL attached to V43C in F-actin became high upon addition of cofilin. •Change of motility of MTSL attached to V43C with cofilin-binding was linear. -- Abstract: Cofilin is an actin-binding protein that promotes F-actin depolymerization. It is well-known that cofilin-coated F-actin is more twisted than naked F-actin, and that the protomer is more tilted. However, the means by which the local changes induced by the binding of individual cofilin proteins proceed to the global conformational changes of the whole F-actin molecule remain unknown. Here we investigated the cofilin-induced changes in several parts of F-actin, through site-directed spin-label electron paramagnetic resonance spectroscopy analyses of recombinant actins containing single reactive cysteines. We found that the global, cooperative conformational changes induced by cofilin-binding, which were detected by the spin-label attached to the Cys374 residue, occurred without the detachment of the D-loop in subdomain 2 from the neighboring protomer. The two processes of local and global changes do not necessarily proceed in sequence.

  3. The Plant-Specific Actin Binding Protein SCAB1 Stabilizes Actin Filaments and Regulates Stomatal Movement in Arabidopsis[C][W

    PubMed Central

    Zhao, Yang; Zhao, Shuangshuang; Mao, Tonglin; Qu, Xiaolu; Cao, Wanhong; Zhang, Li; Zhang, Wei; He, Liu; Li, Sidi; Ren, Sulin; Zhao, Jinfeng; Zhu, Guoli; Huang, Shanjin; Ye, Keqiong; Yuan, Ming; Guo, Yan

    2011-01-01

    Microfilament dynamics play a critical role in regulating stomatal movement; however, the molecular mechanism underlying this process is not well understood. We report here the identification and characterization of STOMATAL CLOSURE-RELATED ACTIN BINDING PROTEIN1 (SCAB1), an Arabidopsis thaliana actin binding protein. Plants lacking SCAB1 were hypersensitive to drought stress and exhibited reduced abscisic acid-, H2O2-, and CaCl2-regulated stomatal movement. In vitro and in vivo analyses revealed that SCAB1 binds, stabilizes, and bundles actin filaments. SCAB1 shares sequence similarity only with plant proteins and contains a previously undiscovered actin binding domain. During stomatal closure, actin filaments switched from a radial orientation in open stomata to a longitudinal orientation in closed stomata. This switch took longer in scab1 plants than in wild-type plants and was correlated with the delay in stomatal closure seen in scab1 mutants in response to drought stress. Our results suggest that SCAB1 is required for the precise regulation of actin filament reorganization during stomatal closure. PMID:21719691

  4. Simiate is an Actin binding protein involved in filopodia dynamics and arborization of neurons

    PubMed Central

    Derlig, Kristin; Ehrhardt, Toni; Gießl, Andreas; Brandstätter, Johann H.; Enz, Ralf; Dahlhaus, Regina

    2014-01-01

    The Actin cytoskeleton constitutes the functional base for a multitude of cellular processes extending from motility and migration to cell mechanics and morphogenesis. The latter is particularly important to neuronal cells since the accurate functioning of the brain crucially depends on the correct arborization of neurons, a process that requires the formation of several dozens to hundreds of dendritic branches. Recently, a model was proposed where different transcription factors are detailed to distinct facets and phases of dendritogenesis and exert their function by acting on the Actin cytoskeleton, however, the proteins involved as well as the underlying molecular mechanisms are largely unknown. Here, we demonstrate that Simiate, a protein previously indicated to activate transcription, directly associates with both, G- and F-Actin and in doing so, affects Actin polymerization and Actin turnover in living cells. Imaging studies illustrate that Simiate particularly influences filopodia dynamics and specifically increases the branching of proximal, but not distal dendrites of developing neurons. The data suggests that Simiate functions as a direct molecular link between transcription regulation on one side, and dendritogenesis on the other, wherein Simiate serves to coordinate the development of proximal and distal dendrites by acting on the Actin cytoskeleton of filopodia and on transcription regulation, hence supporting the novel model. PMID:24782708

  5. Structural and Functional Dissection of the Abp1 ADFH Actin-binding Domain Reveals Versatile In Vivo Adapter Functions

    SciTech Connect

    Quintero-Monzon,O.; Rodal, A.; Strokopytov, B.; Almo, S.; Goode, B.

    2005-01-01

    Abp1 is a multidomain protein that regulates the Arp2/3 complex and links proteins involved in endocytosis to the actin cytoskeleton. All of the proposed cellular functions of Abp1 involve actin filament binding, yet the actin binding site(s) on Abp1 have not been identified, nor has the importance of actin binding for Abp1 localization and function in vivo been tested. Here, we report the crystal structure of the Saccharomyces cerevisiae Abp1 actin-binding actin depolymerizing factor homology (ADFH) domain and dissect its activities by mutagenesis. Abp1-ADFH domain and ADF/cofilin structures are similar, and they use conserved surfaces to bind actin; however, there are also key differences that help explain their differential effects on actin dynamics. Using point mutations, we demonstrate that actin binding is required for localization of Abp1 in vivo, the lethality caused by Abp1 overexpression, and the ability of Abp1 to activate Arp2/3 complex. Furthermore, we genetically uncouple ABP1 functions that overlap with SAC6, SLA1, and SLA2, showing they require distinct combinations of activities and interactions. Together, our data provide the first structural and functional view of the Abp1-actin interaction and show that Abp1 has distinct cellular roles as an adapter, linking different sets of ligands for each function.

  6. Localization of cofilin gene to 1q25

    SciTech Connect

    Hung, W.Y.; Deng, H.X.; Hentati, H.A.

    1994-09-01

    Cofilin is a 21 kD actin-binding protein which has recently been identified as an important intracellular messenger that activates resting T-lymphocytes for clonal growth and expression of their functional repertoires. To determine the chromosomal location of the cofilin gene, a cDNA fragment, 276 bp downstream from initial codon to poly A tail, was used as a probe to screen a human genomic DNA lamda phage library. Four positive phage clones were isolated from 400,000 phage plaques. The size of the genomic inserts ranged from 14 kb to 20 kb. The DNA from these phage clones were labeled with digoxigenin and hybridized to metaphase chromosome preparations. The hybridization signals were detected with sheep anti-digoxigenin and FITC-conjugated rabbit anti-sheep antibodies. Fluorescence signal was amplified once with FITC-conjugated goat anti-rabbit antibody. The results indicate that cofilin gene is located at chromosome 1q25.

  7. Following the Viterbi Path to Deduce Flagellar Actin-Interacting Proteins of Leishmania spp.: Report on Cofilins and Twinfilins

    NASA Astrophysics Data System (ADS)

    Pacheco, Ana Carolina L.; Araújo, Fabiana F.; Kamimura, Michel T.; Medeiros, Sarah R.; Viana, Daniel A.; Oliveira, Fátima de Cássia E.; Filho, Raimundo Araújo; Costa, Marcília P.; Oliveira, Diana M.

    2007-11-01

    For performing vital cellular processes, such as motility, eukaryotic cells rely on the actin cytoskeleton, whose structure and dynamics are tightly controlled by a large number of actin-interacting (AIP) or actin-related/regulating (ARP) proteins. Trypanosomatid protozoa, such as Leishmania, rely on their flagellum for motility and sensory reception, which are believed to allow parasite migration, adhesion, invasion and even persistence on mammalian host tissues to cause disease. Actin can determine cell stiffness and transmit force during mechanotransduction, cytokinesis, cell motility and other cellular shape changes, while the identification and analyses of AIPs can help to improve understanding of their mechanical properties on physiological architectures, such as the present case regarding Leishmania flagellar apparatus. This work conveniently apply bioinformatics tools in some refined pattern recognition techniques (such as hidden Markov models (HMMs) through the Viterbi algorithm/path) in order to improve the recognition of actin-binding/interacting activity through identification of AIPs in genomes, transcriptomes and proteomes of Leishmania species. We here report cofilin and twinfilin as putative components of the flagellar apparatus, a direct bioinformatics contribution in the secondary annotation of Leishmania and trypanosomatid genomes.

  8. Novel functions for ADF/cofilin in excitatory synapses - lessons from gene-targeted mice

    PubMed Central

    Rust, Marco B

    2015-01-01

    Actin filaments (F-actin) are the major structural component of excitatory synapses. In excitatory synapses, F-actin is enriched in presynaptic terminals and in postsynaptic dendritic spines, and actin dynamics – the spatiotemporally controlled assembly and disassembly of F-actin – have been implicated in pre- and postsynaptic physiology, additionally to their function in synapse morphology. Hence, actin binding proteins that control actin dynamics have moved into the focus as regulators of synapse morphology and physiology. Actin depolymerizing proteins of the ADF/cofilin family are important regulators of actin dynamics, and several recent studies highlighted the relevance of cofilin 1 for dendritic spine morphology, trafficking of postsynaptic glutamate receptors, and synaptic plasticity. Conversely, almost nothing was known about the synaptic function of ADF, a second ADF/cofilin family member present at excitatory synapses, and it remained unknown whether ADF/cofilin is relevant for presynaptic physiology. To comprehensively characterize the synaptic function of ADF/cofilin we made use of mutant mice lacking either ADF or cofilin 1 or both proteins. Our analysis revealed presynaptic defects (altered distribution and enhanced exocytosis of synaptic vesicles) and behavioral abnormalities reminiscent of attention deficit-hyperactivity disorder in double mutants that were not present in single mutants. Hence, by exploiting gene-targeted mice, we demonstrated the relevance of ADF for excitatory synapses, and we unraveled novel functions for ADF/cofilin in presynaptic physiology and behavior. PMID:27066177

  9. 65-kilodalton protein phosphorylated by interleukin 2 stimulation bears two putative actin-binding sites and two calcium-binding sites

    SciTech Connect

    Zu, Youli; Shigesada, Katsuya; Hanaoka, Masao; Namba, Yuziro ); Nishida, Eisuke ); Kubota, Ichiro ); Kohno, Michiaki )

    1990-09-11

    The authors have previously characterized a 65-kilodalton protein (p65) as an interleukin 2 stimulated phosphoprotein in human T cells and showed that three endopeptide sequences of p65 are present in the sequence of l-plastin. In this paper, they present the complete primary structure of p65 based on the cDNA isolated from a human T lymphocyte (KUT-2) cDNA library. Analysis of p65 sequences and the amino acid composition of cleaved p65 N-terminal peptide indicated that the deduced p65 amino acid sequence exactly coincides with that of l-plastin over the C-terminal 580 residues and has a 57-residue extension at the N-terminus to l-plastin. Computer-assisted structural analysis revealed that p65 is a multidomain molecule involving at least three intriguing functional domains: two putative calcium-binding sites along the N-terminal 80 amino acid residues; a putative calmodulin-binding site following the calcium-binding region; and two tandem repeats of putative actin-binding domains in its middle and C-terminal parts, each containing approximately 240 amino acid residues. These results suggest that p65 belongs to actin-binding proteins.

  10. Linking microfilaments to intracellular membranes: the actin-binding and vesicle-associated protein comitin exhibits a mannose-specific lectin activity.

    PubMed Central

    Jung, E; Fucini, P; Stewart, M; Noegel, A A; Schleicher, M

    1996-01-01

    Comitin is a 24 kDa actin-binding protein from Dictyostelium discoideum that is located primarily on Golgi and vesicle membranes. We have probed the molecular basis of comitin's interaction with both actin and membranes using a series of truncation mutants obtained by expressing the appropriate cDNA in Escherichia coli. Comitin dimerizes in solution; its principle actin-binding activity is located between residues 90 and 135. The N-terminal 135 'core' residues of comitin contain a 3-fold sequence repeat that is homologous to several monocotyledon lectins and which retains key residues that determine these lectins' three-dimensional structure and mannose binding. These repeats of comitin appear to mediate its interaction with mannose residues in glycoproteins or glycolipids on the cytoplasmic surface of membrane vesicles from D.discoideum, and comitin can be released from membranes with mannose. Our data indicate that comitin binds to vesicle membranes via mannose residues and, by way of its interaction with actin, links these membranes to the cytoskeleton. Images PMID:8635456

  11. Microtubule-associated Protein 2c Reorganizes Both Microtubules and Microfilaments into Distinct Cytological Structures in an Actin-binding Protein-280–deficient Melanoma Cell Line

    PubMed Central

    Cunningham, C. Casey; Leclerc, Nicole; Flanagan, Lisa A.; Lu, Mei; Janmey, Paul A.; Kosik, Kenneth S.

    1997-01-01

    The emergence of processes from cells often involves interactions between microtubules and microfilaments. Interactions between these two cytoskeletal systems are particularly apparent in neuronal growth cones. The juvenile isoform of the neuronal microtubule-associated protein 2 (MAP2c) is present in growth cones, where we hypothesize it mediates interactions between microfilaments and microtubules. To approach this problem in vivo, we used the human melanoma cell, M2, which lacks actin-binding protein-280 (ABP-280) and forms membrane blebs, which are not seen in wild-type or ABP-transfected cells. The microinjection of tau or mature MAP2 rescued the blebbing phenotype; MAP2c not only caused cessation of blebbing but also induced the formation of two distinct cellular structures. These were actin-rich lamellae, which often included membrane ruffles, and microtubule-bearing processes. The lamellae collapsed after treatment with cytochalasin D, and the processes retracted after treatment with colchicine. MAP2c was immunocytochemically visualized in zones of the cell that were devoid of tubulin, such as regions within the lamellae and in association with membrane ruffles. In vitro rheometry confirmed that MAP2c is an efficient actin gelation protein capable of organizing actin filaments into an isotropic array at very low concentrations; tau and mature MAP2 do not share this rheologic property. These results suggest that MAP2c engages in functionally specific interactions not only with microtubules but also with microfilaments. PMID:9049250

  12. Transgenic Expression of the Formin Protein Fhod3 Selectively in the Embryonic Heart: Role of Actin-Binding Activity of Fhod3 and Its Sarcomeric Localization during Myofibrillogenesis

    PubMed Central

    Fujimoto, Noriko; Kan-o, Meikun; Ushijima, Tomoki; Kage, Yohko; Tominaga, Ryuji; Sumimoto, Hideki; Takeya, Ryu

    2016-01-01

    Fhod3 is a cardiac member of the formin family proteins that play pivotal roles in actin filament assembly in various cellular contexts. The targeted deletion of mouse Fhod3 gene leads to defects in cardiogenesis, particularly during myofibrillogenesis, followed by lethality at embryonic day (E) 11.5. However, it remains largely unknown how Fhod3 functions during myofibrillogenesis. In this study, to assess the mechanism whereby Fhod3 regulates myofibrillogenesis during embryonic cardiogenesis, we generated transgenic mice expressing Fhod3 selectively in embryonic cardiomyocytes under the control of the β-myosin heavy chain (MHC) promoter. Mice expressing wild-type Fhod3 in embryonic cardiomyocytes survive to adulthood and are fertile, whereas those expressing Fhod3 (I1127A) defective in binding to actin die by E11.5 with cardiac defects. This cardiac phenotype of the Fhod3 mutant embryos is almost identical to that observed in Fhod3 null embryos, suggesting that the actin-binding activity of Fhod3 is crucial for embryonic cardiogenesis. On the other hand, the β-MHC promoter-driven expression of wild-type Fhod3 sufficiently rescues cardiac defects of Fhod3-null embryos, indicating that the Fhod3 protein expressed in a transgenic manner can function properly to achieve myofibril maturation in embryonic cardiomyocytes. Using the transgenic mice, we further examined detailed localization of Fhod3 during myofibrillogenesis in situ and found that Fhod3 localizes to the specific central region of nascent sarcomeres prior to massive rearrangement of actin filaments and remains there throughout myofibrillogenesis. Taken together, the present findings suggest that, during embryonic cardiogenesis, Fhod3 functions as the essential reorganizer of actin filaments at the central region of maturating sarcomeres via the actin-binding activity of the FH2 domain. PMID:26848968

  13. Cofilin contributes to phagocytosis of IgG-opsonized particles but not non-opsonized particles in RAW264 macrophages.

    PubMed

    Lu, Yanmeng; Cao, Lei; Egami, Youhei; Kawai, Katsuhisa; Araki, Nobukazu

    2016-06-01

    Cofilin is an actin-binding protein that severs actin filaments. It plays a key role in regulating actin cytoskeletal remodeling, thereby contributing to diverse cellular functions. However, the involvement of cofilin in phagocytosis remains to be elucidated. We examined the spatiotemporal localization of cofilin during phagocytosis of IgG-opsonized erythrocytes, IgG-opsonized latex beads and non-opsonized latex beads. Live-cell imaging showed that GFP-cofilin accumulates in the sites of IgG-opsonized particle binding and in phagocytic cups. Moreover, immunofluorescence microscopy revealed that endogenous cofilin localizes to phagocytic cups engulfing IgG-opsonized particles, but not non-opsonized latex beads. Scanning electron microscopy demonstrated a notable difference in morphology between phagocytic structures in IgG-dependent and IgG-independent phagocytosis. In phagocytosis of IgG-opsonized particles, sheet-like pseudopodia extended along the surface of IgG-opsonized particles to form phagocytic cups. In contrast, in opsonin-independent phagocytosis, long finger-like filopodia captured non-opsonized latex beads. Importantly, non-opsonized beads sank into the cells without extending phagocytic cups. Our analysis of cofilin mutant expression demonstrates that phagocytosis of IgG-opsonized particles is enhanced in cells expressing wild-type cofilin or active mutant cofilin-S3A, whereas the uptake of non-opsonized latex beads is not. These data suggest that cofilin promotes actin cytoskeletal remodeling to form phagocytic cups by accelerating actin turnover and thereby facilitating phagosome formation. In contrast, cofilin is not involved in opsonin-independent phagocytosis of latex beads. PMID:26754560

  14. The 43-K protein, v1, associated with acetylcholine receptor containing membrane fragments is an actin-binding protein.

    PubMed Central

    Walker, J H; Boustead, C M; Witzemann, V

    1984-01-01

    Acetylcholine receptor enriched membrane fragments were obtained from the electric organs of Torpedo marmorata. The purified membrane fragments contained several proteins in addition to the acetylcholine receptor subunits. One of these was shown to be actin by means of immune blotting with a monoclonal antibody. Brief treatment of the membranes with pH 11.0 buffer removed actin and the other non-receptor proteins including the receptor-associated 43 000 mol. wt. polypeptide. This polypeptide was shown to bind actin after transferring the proteins from one- and two-dimensional polyacrylamide gels to nitrocellulose paper and incubating the nitrocellulose blots with actin. Specifically bound actin was demonstrated using the monoclonal antibodies to actin. No calcium or calmodulin dependency of binding was observed. The findings suggest that the 43 000 mol. wt. polypeptide is a link between the membrane-bound acetylcholine receptor and the cytoskeleton. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. PMID:6389118

  15. Activated ADF/cofilin sequesters phosphorylated microtubule-associated-protein during the assembly of Alzheimer-like neuritic cytoskeletal striations

    PubMed Central

    Whiteman, Ineka T.; Gervasio, Othon L.; Cullen, Karen M.; Guillemin, Gilles J.; Jeong, Erica V.; Witting, Paul K.; Antao, Shane T.; Minamide, Laurie S.; Bamburg, James R.; Goldsbury, Claire

    2009-01-01

    In Alzheimer disease (AD), rod-like cofilin aggregates (cofilin-actin rods) and thread-like inclusions containing phosphorylated microtubule-associated protein (pMAP) tau form in the brain (neuropil threads) and the extent of their presence correlates with cognitive decline and disease progression. The assembly mechanism of these respective pathological lesions and the relationship between them is poorly understood, yet vital to understanding the causes of sporadic AD. We demonstrate that during mitochondrial inhibition, activated actin-depolymerizing factor (ADF)/cofilin assemble into rods along processes of cultured primary neurons that recruit pMAP/tau and mimic neuropil threads. Fluorescence Resonance Energy Transfer (FRET) analysis revealed co-localization of cofilin-GFP and pMAP in rods, suggesting their close proximity within a cytoskeletal inclusion complex. The relationship between pMAP and cofilin-actin rods was further investigated using actin-modifying drugs and siRNA knockdown of ADF/cofilin in primary neurons. The results suggest that activation of ADF/cofilin and generation of cofilin-actin rods is required for the subsequent recruitment of pMAP into the inclusions. Additionally we were able to induce the formation of pMAP-positive ADF/cofilin rods by exposing cells to exogenous Aβ peptides. These results reveal a common pathway for pMAP and cofilin accumulation in neuronal processes. The requirement of activated ADF/cofilin for the sequestration of pMAP suggests that neuropil thread structures in the AD brain may be initiated by elevated cofilin activation and F-actin bundling that can be caused by oxidative stress, mitochondrial dysfunction or Aβ peptides, all suspected initiators of synaptic loss and neurodegeneration in AD. PMID:19828813

  16. Interaptin, an Actin-binding Protein of the α-Actinin Superfamily in Dictyostelium discoideum, Is Developmentally and cAMP-regulated and Associates with Intracellular Membrane Compartments

    PubMed Central

    Rivero, Francisco; Kuspa, Adam; Brokamp, Regine; Matzner, Monika; Noegel, Angelika A.

    1998-01-01

    In a search for novel members of the α-actinin superfamily, a Dictyostelium discoideum genomic library in yeast artificial chromosomes (YAC) was screened under low stringency conditions using the acting-binding domain of the gelation factor as probe. A new locus was identified and 8.6 kb of genomic DNA were sequenced that encompassed the whole abpD gene. The DNA sequence predicts a protein, interaptin, with a calculated molecular mass of 204,300 D that is constituted by an actin-binding domain, a central coiled-coil rod domain and a membrane-associated domain. In Northern blot analyses a cAMP-stimulated transcript of 5.8 kb is expressed at the stage when cell differentiation occurs. Monoclonal antibodies raised against bacterially expressed interaptin polypeptides recognized a 200-kD developmentally and cAMP-regulated protein and a 160-kD constitutively expressed protein in Western blots. In multicellular structures, interaptin appears to be enriched in anterior-like cells which sort to the upper and lower cups during culmination. The protein is located at the nuclear envelope and ER. In mutants deficient in interaptin development is delayed, but the morphology of the mature fruiting bodies appears normal. When starved in suspension abpD− cells form EDTA-stable aggregates, which, in contrast to wild type, dissociate. Based on its domains and location, interaptin constitutes a potential link between intracellular membrane compartments and the actin cytoskeleton. PMID:9700162

  17. Cofilin Oligomer Formation Occurs In Vivo and Is Regulated by Cofilin Phosphorylation

    PubMed Central

    Goyal, Pankaj; Pandey, Dharmendra; Brünnert, Daniela; Hammer, Elke; Zygmunt, Marek; Siess, Wolfgang

    2013-01-01

    Background ADF/cofilin proteins are key regulators of actin dynamics. Their function is inhibited by LIMK-mediated phosphorylation at Ser-3. Previous in vitro studies have shown that dependent on its concentration, cofilin either depolymerizes F-actin (at low cofilin concentrations) or promotes actin polymerization (at high cofilin concentrations). Methodology/Principal Findings We found that after in vivo cross-linking with different probes, a cofilin oligomer (65 kDa) could be detected in platelets and endothelial cells. The cofilin oligomer did not contain actin. Notably, ADF that only depolymerizes F-actin was present mainly in monomeric form. Furthermore, we found that formation of the cofilin oligomer is regulated by Ser-3 cofilin phosphorylation. Cofilin but not phosphorylated cofilin was present in the endogenous cofilin oligomer. In vitro, formation of cofilin oligomers was drastically reduced after phosphorylation by LIMK2. In endothelial cells, LIMK-mediated cofilin phosphorylation after thrombin-stimulation of EGFP- or DsRed2-tagged cofilin transfected cells reduced cofilin aggregate formation, whereas inhibition of cofilin phosphorylation after Rho-kinase inhibitor (Y27632) treatment of endothelial cells promoted formation of cofilin aggregates. In platelets, cofilin dephosphorylation after thrombin-stimulation and Y27632 treatment led to an increased formation of the cofilin oligomer. Conclusion/Significance Based on our results, we propose that an equilibrium exists between the monomeric and oligomeric forms of cofilin in intact cells that is regulated by cofilin phosphorylation. Cofilin phosphorylation at Ser-3 may induce conformational changes on the protein-protein interacting surface of the cofilin oligomer, thereby preventing and/or disrupting cofilin oligomer formation. Cofilin oligomerization might explain the dual action of cofilin on actin dynamics in vivo. PMID:23951242

  18. Identification of regions within the Legionella pneumophila VipA effector protein involved in actin binding and polymerization and in interference with eukaryotic organelle trafficking.

    PubMed

    Bugalhão, Joana N; Mota, Luís Jaime; Franco, Irina S

    2016-02-01

    The Legionella pneumophila effector protein VipA is an actin nucleator that co-localizes with actin filaments and early endosomes in infected macrophages and which interferes with organelle trafficking when expressed in yeast. To identify the regions of VipA involved in its subcellular localization and functions, we ectopically expressed specific VipA mutant proteins in eukaryotic cells. This indicated that the characteristic punctate distribution of VipA depends on its NH2 -terminal (amino acid residues 1-133) and central coiled-coil (amino acid residues 133-206) regions, and suggested a role for the COOH-terminal (amino acid residues 206-339) region in association with actin filaments and for the NH2 -terminal in co-localization with early endosomes. Co-immunoprecipitation and in vitro assays showed that the COOH-terminal region of VipA is necessary and sufficient to mediate actin binding, and is essential but insufficient to induce microfilament formation. Assays in yeast revealed that the NH2 and the COOH-terminal regions, and possibly an NPY motif within the NH2 region of VipA, are necessary for interference with organelle trafficking. Overall, this suggests that subversion of eukaryotic vesicular trafficking by VipA involves both its ability to associate with early endosomes via its NH2 -terminal region and its capacity to bind and polymerize actin through its COOH-terminal region. PMID:26626407

  19. Cyclic stretch promotes osteogenesis-related gene expression in osteoblast-like cells through a cofilin-associated mechanism

    PubMed Central

    GAO, JIE; FU, SHANMIN; ZENG, ZHAOBIN; LI, FEIFEI; NIU, QIANNAN; JING, DA; FENG, XUE

    2016-01-01

    Osteoblasts have the capacity to perceive and transduce mechanical signals, and thus, regulate the mRNA and protein expression of a variety of genes associated with osteogenesis. Cytoskeletal reconstruction, as one of the earliest perception events for external mechanical stimulation, has previously been demonstrated to be essential for mechanotransduction in bone cells. However, the mechanism by which mechanical signals induce cytoskeletal deformation remains poorly understood. The actin-binding protein, cofilin, promotes the depolymerization of actin and is understood to be important in the regulation of activities in various cell types, including endothelial, neuronal and muscle cells. However, to the best of our knowledge, the importance of cofilin in osteoblastic mechanotransduction has not been previously investigated. In the present study, osteoblast-like MG-63 cells were subjected to physiological cyclic stretch stimulation (12% elongation) for 1, 4, 8, 12 and 24 h, and the expression levels of cofilin and osteogenesis-associated genes were quantified with reverse transcription-quantitative polymerase chain reaction, immunofluorescence staining and western blotting analyses. Additionally, knockdown of cofilin using RNA interference was conducted, and the mRNA levels of osteogenesis-associated genes were compared between osteoblast-like cells in the presence and absence of cofilin gene knockdown. The results of the present study demonstrated that cyclic stretch stimulates the expression of genes associated with osteoblastic activities in MG-63 cells, including alkaline phosphatase (ALP), osteocalcin (OCN), runt-related transcription factor 2 (Runx2) and collagen-1 (COL-1). Cyclic stretch also regulates the mRNA and protein expression of cofilin in MG-63 cells. Furthermore, stretch-induced increases in the levels of osteogenesis-associated genes, including ALP, OCN, Runx2 and COL-1, were reduced following cofilin gene knockdown. Together, these results

  20. Arabidopsis CROLIN1, a Novel Plant Actin-binding Protein, Functions in Cross-linking and Stabilizing Actin Filaments*

    PubMed Central

    Jia, Honglei; Li, Jisheng; Zhu, Jingen; Fan, Tingting; Qian, Dong; Zhou, Yuelong; Wang, Jiaojiao; Ren, Haiyun; Xiang, Yun; An, Lizhe

    2013-01-01

    Higher order actin filament structures are necessary for cytoplasmic streaming, organelle movement, and other physiological processes. However, the mechanism by which the higher order cytoskeleton is formed in plants remains unknown. In this study, we identified a novel actin-cross-linking protein family (named CROLIN) that is well conserved only in the plant kingdom. There are six isovariants of CROLIN in the Arabidopsis genome, with CROLIN1 specifically expressed in pollen. In vitro biochemical analyses showed that CROLIN1 is a novel actin-cross-linking protein with binding and stabilizing activities. Remarkably, CROLIN1 can cross-link actin bundles into actin networks. CROLIN1 loss of function induces pollen germination and pollen tube growth hypersensitive to latrunculin B. All of these results demonstrate that CROLIN1 may play an important role in stabilizing and remodeling actin filaments by binding to and cross-linking actin filaments. PMID:24072702

  1. Actin-binding protein alpha-actinin 4 (ACTN4) is a transcriptional co-activator of RelA/p65 sub-unit of NF-kB

    PubMed Central

    Aksenova, Vasilisa; Turoverova, Lidia; Khotin, Mikhail; Magnusson, Karl-Eric; Tulchinsky, Eugene; Melino, Gerry; Pinaev, George P.; Barlev, Nickolai; Tentler, Dmitri

    2013-01-01

    ACTN4 is an actin-binding protein that participates in cytoskeleton organisation. It resides both in the cytoplasm and nucleus and physically associates with various transcription factors. Here, we describe an effect of ACTN4 expression on transcriptional activity of the RelA/p65 subunit of NF-kB. We demonstrate that ACTN4 enhances RelA/p65-dependant expression of c-fos, MMP-3 and MMP-1 genes, but it does not affect TNC, ICAM1 and FN1 expression. Importantly, actin-binding domains of ACTN4 are not critical for the nuclear translocation and co-activation of RelA/p65-dependent transcription. Collectively, our data suggest that in the nucleus, ACTN4 functions as a selective transcriptional co-activator of RelA/p65. PMID:23482348

  2. The actin-binding protein EPS8 binds VE-cadherin and modulates YAP localization and signaling

    PubMed Central

    Disanza, Andrea; Bravi, Luca; Barrios-Rodiles, Miriam; Corada, Monica; Frittoli, Emanuela; Savorani, Cecilia; Lampugnani, Maria Grazia; Boggetti, Barbara; Niessen, Carien; Wrana, Jeff L.

    2015-01-01

    Vascular endothelial (VE)–cadherin transfers intracellular signals contributing to vascular hemostasis. Signaling through VE-cadherin requires association and activity of different intracellular partners. Yes-associated protein (YAP)/TAZ transcriptional cofactors are important regulators of cell growth and organ size. We show that EPS8, a signaling adapter regulating actin dynamics, is a novel partner of VE-cadherin and is able to modulate YAP activity. By biochemical and imaging approaches, we demonstrate that EPS8 associates with the VE-cadherin complex of remodeling junctions promoting YAP translocation to the nucleus and transcriptional activation. Conversely, in stabilized junctions, 14–3-3–YAP associates with the VE–cadherin complex, whereas Eps8 is excluded. Junctional association of YAP inhibits nuclear translocation and inactivates its transcriptional activity both in vitro and in vivo in Eps8-null mice. The absence of Eps8 also increases vascular permeability in vivo, but did not induce other major vascular defects. Collectively, we identified novel components of the adherens junction complex, and we introduce a novel molecular mechanism through which the VE-cadherin complex controls YAP transcriptional activity. PMID:26668327

  3. Identification and characterization of espin, an actin-binding protein localized to the F-actin-rich junctional plaques of Sertoli cell ectoplasmic specializations.

    PubMed

    Bartles, J R; Wierda, A; Zheng, L

    1996-06-01

    Ectoplasmic specializations are membrane-cytoskeletal assemblages found in Sertoli cells at sites of attachment to elongate spermatids or neighboring Sertoli cells. They are characterized in part by the presence of a unique junctional plaque which contains a narrow layer of parallel actin bundles sandwiched between the Sertoli cell plasma membrane and an affiliated cistern of endoplasmic reticulum. Using a monoclonal antibody, we have identified 'espin,' a novel actin-binding protein localized to ectoplasmic specializations. By immunogold electron microscopy, espin was localized to the parallel actin bundles of ectoplasmic specializations at sites where Sertoli cells contacted the heads of elongate spermatids. The protein was also detected at the sites of ectoplasmic specializations between neighboring Sertoli cells. Espin exhibits an apparent molecular mass of approximately 110 kDa in SDS gels. It is encoded by an approximately 2.9 kb mRNA, which was found to be specific to testis among the 11 rat organs and tissues examined. On the basis of cDNA sequence, espin is predicted to be an 836 amino acid protein which contains 8 ankyrin-like repeats in its N-terminal third, a potential P-loop, two proline-rich peptides and two peptides which contain clusters of multiple glutamates bracketed by arginines, lysines and glutamines in a pattern reminiscent of the repetitive motif found in the protein trichohyalin. The ankyrin-like repeats and a 66 amino acid peptide in the C terminus show significant sequence similarity to proteins encoded by the forked gene of Drosophila. A fusion protein containing the C-terminal 378 amino acids of espin was found to bind with high affinity (Kd = approximately 10 nM) to F-actin in vitro with a stoichiometry of approximately 1 espin per 6 actin monomers. When expressed by transfected NRK fibroblasts, the same C-terminal fragment of espin was observed to decorate actin fibers or cables. On the basis of its structure, localization and

  4. Phosphatidylinositol 3-Kinase-Associated Protein (PI3KAP)/XB130 Crosslinks Actin Filaments through Its Actin Binding and Multimerization Properties In Vitro and Enhances Endocytosis in HEK293 Cells.

    PubMed

    Yamanaka, Daisuke; Akama, Takeshi; Chida, Kazuhiro; Minami, Shiro; Ito, Koichi; Hakuno, Fumihiko; Takahashi, Shin-Ichiro

    2016-01-01

    Actin-crosslinking proteins control actin filament networks and bundles and contribute to various cellular functions including regulation of cell migration, cell morphology, and endocytosis. Phosphatidylinositol 3-kinase-associated protein (PI3KAP)/XB130 has been reported to be localized to actin filaments (F-actin) and required for cell migration in thyroid carcinoma cells. Here, we show a role for PI3KAP/XB130 as an actin-crosslinking protein. First, we found that the carboxyl terminal region of PI3KAP/XB130 containing amino acid residues 830-840 was required and sufficient for localization to F-actin in NIH3T3 cells, and this region is directly bound to F-actin in vitro. Moreover, actin-crosslinking assay revealed that recombinant PI3KAP/XB130 crosslinked F-actin. In general, actin-crosslinking proteins often multimerize to assemble multiple actin-binding sites. We then investigated whether PI3KAP/XB130 could form a multimer. Blue native-PAGE analysis showed that recombinant PI3KAP/XB130 was detected at 250-1200 kDa although the molecular mass was approximately 125 kDa, suggesting that PI3KAP/XB130 formed multimers. Furthermore, we found that the amino terminal 40 amino acids were required for this multimerization by co-immunoprecipitation assay in HEK293T cells. Deletion mutants of PI3KAP/XB130 lacking the actin-binding region or the multimerizing region did not crosslink actin filaments, indicating that actin binding and multimerization of PI3KAP/XB130 were necessary to crosslink F-actin. Finally, we examined roles of PI3KAP/XB130 on endocytosis, an actin-related biological process. Overexpression of PI3KAP/XB130 enhanced dextran uptake in HEK 293 cells. However, most of the cells transfected with the deletion mutant lacking the actin-binding region incorporated dextran to a similar extent as control cells. Taken together, these results demonstrate that PI3KAP/XB130 crosslinks F-actin through both its actin-binding region and multimerizing region and plays

  5. Phosphatidylinositol 3-Kinase-Associated Protein (PI3KAP)/XB130 Crosslinks Actin Filaments through Its Actin Binding and Multimerization Properties In Vitro and Enhances Endocytosis in HEK293 Cells

    PubMed Central

    Yamanaka, Daisuke; Akama, Takeshi; Chida, Kazuhiro; Minami, Shiro; Ito, Koichi; Hakuno, Fumihiko; Takahashi, Shin-Ichiro

    2016-01-01

    Actin-crosslinking proteins control actin filament networks and bundles and contribute to various cellular functions including regulation of cell migration, cell morphology, and endocytosis. Phosphatidylinositol 3-kinase-associated protein (PI3KAP)/XB130 has been reported to be localized to actin filaments (F-actin) and required for cell migration in thyroid carcinoma cells. Here, we show a role for PI3KAP/XB130 as an actin-crosslinking protein. First, we found that the carboxyl terminal region of PI3KAP/XB130 containing amino acid residues 830–840 was required and sufficient for localization to F-actin in NIH3T3 cells, and this region is directly bound to F-actin in vitro. Moreover, actin-crosslinking assay revealed that recombinant PI3KAP/XB130 crosslinked F-actin. In general, actin-crosslinking proteins often multimerize to assemble multiple actin-binding sites. We then investigated whether PI3KAP/XB130 could form a multimer. Blue native-PAGE analysis showed that recombinant PI3KAP/XB130 was detected at 250–1200 kDa although the molecular mass was approximately 125 kDa, suggesting that PI3KAP/XB130 formed multimers. Furthermore, we found that the amino terminal 40 amino acids were required for this multimerization by co-immunoprecipitation assay in HEK293T cells. Deletion mutants of PI3KAP/XB130 lacking the actin-binding region or the multimerizing region did not crosslink actin filaments, indicating that actin binding and multimerization of PI3KAP/XB130 were necessary to crosslink F-actin. Finally, we examined roles of PI3KAP/XB130 on endocytosis, an actin-related biological process. Overexpression of PI3KAP/XB130 enhanced dextran uptake in HEK 293 cells. However, most of the cells transfected with the deletion mutant lacking the actin-binding region incorporated dextran to a similar extent as control cells. Taken together, these results demonstrate that PI3KAP/XB130 crosslinks F-actin through both its actin-binding region and multimerizing region and

  6. Luteinizing Hormone Receptor-Stimulated Progesterone Production by Preovulatory Granulosa Cells Requires Protein Kinase A-Dependent Activation/Dephosphorylation of the Actin Dynamizing Protein Cofilin

    PubMed Central

    Karlsson, Amelia B.; Maizels, Evelyn T.; Flynn, Maxfield P.; Jones, Jonathan C.; Shelden, Eric A.; Bamburg, James R.; Hunzicker-Dunn, Mary

    2010-01-01

    Activation of the LH receptor (LHR) on preovulatory granulosa cells stimulates the cAMP/protein kinase A (PKA) pathway to regulate expression of genes required for ovulation and luteinization. LHR signaling also initiates rearrangement of the actin cytoskeleton. Because disruption of the actin cytoskeleton has been causally linked to steroidogenesis in various cell models, we sought to identify the cellular mechanisms that may modulate reorganization of the actin cytoskeleton and to determine whether cytoskeletal reorganization is required for steroidogenesis. Herein we report that LHR signaling in preovulatory granulosa cells promotes rapid dephosphorylation of the actin-depolymerizing factor cofilin at Ser3 that is dependent on PKA. The LHR-stimulated dephosphorylation of cofilin(Ser3) switches on cofilin activity to bind actin filaments and enhance their dynamics. Basal phosphorylation of cofilin(Ser3) is mediated by active/GTP-bound Rho and downstream protein kinases; LHR signaling promotes a decrease in active/GTP-bound Rho by a PKA-dependent mechanism. LHR-dependent Rho inactivation and subsequent activation of cofilin does not involve ERK, epidermal growth factor receptor, or phosphatidylinositol 3-kinase pathways downstream of PKA. To understand the biological significance of cofilin activation, preovulatory granulosa cells were transduced with a mutant cofilin adenoviral vector in which Ser3 was mutated to Glu (S-E cofilin). Inactive S-E cofilin abolished LHR-mediated reorganization of the actin cytoskeleton and caused a 70% decrease in LHR-stimulated progesterone that is obligatory for ovulation. Taken together, these results show that LHR signaling via PKA activates a cofilin-regulated rearrangement of the actin cytoskeleton and that active cofilin is required to initiate progesterone secretion by preovulatory granulosa cells. PMID:20610540

  7. Gcn1 and Actin Binding to Yih1

    PubMed Central

    Sattlegger, Evelyn; Barbosa, João A. R. G.; Moraes, Maria Carolina S.; Martins, Rafael M.; Hinnebusch, Alan G.; Castilho, Beatriz A.

    2011-01-01

    Yeast Yih1 protein and its mammalian ortholog IMPACT, abundant in neurons, are inhibitors of Gcn2, a kinase involved in amino acid homeostasis, stress response, and memory formation. Like Gcn2, Yih1/IMPACT harbors an N-terminal RWD domain that mediates binding to the Gcn2 activator Gcn1. Yih1 competes with Gcn2 for Gcn1 binding, thus inhibiting Gcn2. Yih1 also binds G-actin. Here, we show that Yih1-actin interaction is independent of Gcn1 and that Yih1-Gcn1 binding does not require actin. The Yih1 RWD (residues 1–132) was sufficient for Gcn2 inhibition and Gcn1 binding, but not for actin binding, showing that actin binding is dispensable for inhibiting Gcn2. Actin binding required Yih1 residues 68–258, encompassing part of the RWD and the C-terminal “ancient domain”; however, residues Asp-102 and Glu-106 in helix3 of the RWD were essential for Gcn1 binding and Gcn2 inhibition but dispensable for actin binding. Thus, the Gcn1- and actin-binding sites overlap in the RWD but have distinct binding determinants. Unexpectedly, Yih1 segment 68–258 was defective for inhibiting Gcn2 even though it binds Gcn1 at higher levels than does full-length Yih1. This and other results suggest that Yih1 binds with different requirements to distinct populations of Gcn1 molecules, and its ability to disrupt Gcn1-Gcn2 complexes is dependent on a complete RWD and hindered by actin binding. Modeling of the ancient domain on the bacterial protein YigZ showed peculiarities to the eukaryotic and prokaryotic lineages, suggesting binding sites for conserved cellular components. Our results support a role for Yih1 in a cross-talk between the cytoskeleton and translation. PMID:21239490

  8. ADF/cofilin-actin rods in neurodegenerative diseases

    PubMed Central

    Bamburg, J.R.; Bernstein, B.W.; Davis, R.C.; Flynn, K.C.; Goldsbury, C.; Jensen, J.R.; Maloney, M.T.; Marsden, I.T.; Minamide, L.S.; Pak, C.W.; Shaw, A.E.; Whiteman, I.; Wiggan, O.

    2015-01-01

    Dephosphorylation (activation) of cofilin, an actin binding protein, is stimulated by initiators of neuronal dysfunction and degeneration including oxidative stress, excitotoxic glutamate, ischemia, and soluble forms of β-amyloid peptide (Aβ). Hyperactive cofilin forms rod-shaped cofilin-saturated actin filament bundles (rods). Other proteins are recruited to rods but are not necessary for rod formation. Neuronal cytoplasmic rods accumulate within neurites where they disrupt synaptic function and are a likely cause of synaptic loss without neuronal loss, as occurs early in dementias. Different rod-inducing stimuli target distinct neuronal populations within the hippocampus. Rods form rapidly, often in tandem arrays, in response to stress. They accumulate phosphorylated tau that immunostains for epitopes present in “striated neuropil threads,” characteristic of tau pathology in Alzheimer disease (AD) brain. Thus, rods might aid in further tau modifications or assembly into paired helical filaments, the major component of neurofibrillary tangles (NFTs). Rods can occlude neurites and block vesicle transport. Some rod-inducing treatments cause an increase in secreted Aβ. Thus rods may mediate the loss of synapses, production of excess Aβ, and formation of NFTs, all of the pathological hallmarks of AD. Cofilin-actin rods also form within the nucleus of heat-shocked neurons and are cleared from cells expressing wild type huntingtin protein but not in cells expressing mutant or silenced huntingtin, suggesting a role for nuclear rods in Huntington disease (HD). As an early event in the neurodegenerative cascade, rod formation is an ideal target for therapeutic intervention that might be useful in treatment of many different neurological diseases. PMID:20088812

  9. Immunological Responses and Actin Dynamics in Macrophages Are Controlled by N-Cofilin but Are Independent from ADF

    PubMed Central

    Jönsson, Friederike; Gurniak, Christine B.; Fleischer, Bernhard; Kirfel, Gregor; Witke, Walter

    2012-01-01

    Dynamic changes in the actin cytoskeleton are essential for immune cell function and a number of immune deficiencies have been linked to mutations, which disturb the actin cytoskeleton. In macrophages and dendritic cells, actin remodelling is critical for motility, phagocytosis and antigen presentation, however the actin binding proteins, which control antigen presentation have been poorly characterized. Here we dissect the specific roles of the family of ADF/cofilin F-actin depolymerizing factors in macrophages and in local immune responses. Macrophage migration, cell polarization and antigen presentation to T-cells require n-cofilin mediated F-actin remodelling. Using a conditional mouse model, we show that n-cofilin also controls MHC class II-dependent antigen presentation. Other cellular processes such as phagocytosis and antigen processing were found to be independent of n-cofilin. Our data identify n-cofilin as a novel regulator of antigen presentation, while ADF on the other hand is dispensable for macrophage motility and antigen presentation. PMID:22558315

  10. Arabidopsis AtADF1 is functionally affected by mutations on actin binding sites.

    PubMed

    Dong, Chun-Hai; Tang, Wei-Ping; Liu, Jia-Yao

    2013-03-01

    The plant actin depolymerizing factor (ADF) binds to both monomeric and filamentous actin, and is directly involved in the depolymerization of actin filaments. To better understand the actin binding sites of the Arabidopsis thaliana L. AtADF1, we generated mutants of AtADF1 and investigated their functions in vitro and in vivo. Analysis of mutants harboring amino acid substitutions revealed that charged residues (Arg98 and Lys100) located at the α-helix 3 and forming an actin binding site together with the N-terminus are essential for both G- and F-actin binding. The basic residues on the β-strand 5 (K82/A) and the α-helix 4 (R135/A, R137/A) form another actin binding site that is important for F-actin binding. Using transient expression of CFP-tagged AtADF1 mutant proteins in onion (Allium cepa) peel epidermal cells and transgenic Arabidopsis thaliana L. plants overexpressing these mutants, we analyzed how these mutant proteins regulate actin organization and affect seedling growth. Our results show that the ADF mutants with a lower affinity for actin filament binding can still be functional, unless the affinity for actin monomers is also affected. The G-actin binding activity of the ADF plays an essential role in actin binding, depolymerization of actin polymers, and therefore in the control of actin organization. PMID:23190411

  11. Cellular functions of the ADF/cofilin family at a glance.

    PubMed

    Kanellos, Georgios; Frame, Margaret C

    2016-09-01

    The actin depolymerizing factor (ADF)/cofilin family comprises small actin-binding proteins with crucial roles in development, tissue homeostasis and disease. They are best known for their roles in regulating actin dynamics by promoting actin treadmilling and thereby driving membrane protrusion and cell motility. However, recent discoveries have increased our understanding of the functions of these proteins beyond their well-characterized roles. This Cell Science at a Glance article and the accompanying poster serve as an introduction to the diverse roles of the ADF/cofilin family in cells. The first part of the article summarizes their actions in actin treadmilling and the main mechanisms for their intracellular regulation; the second part aims to provide an outline of the emerging cellular roles attributed to the ADF/cofilin family, besides their actions in actin turnover. The latter part discusses an array of diverse processes, which include regulation of intracellular contractility, maintenance of nuclear integrity, transcriptional regulation, nuclear actin monomer transfer, apoptosis and lipid metabolism. Some of these could, of course, be indirect consequences of actin treadmilling functions, and this is discussed. PMID:27505888

  12. Genome-wide RNAi screen for nuclear actin reveals a network of cofilin regulators

    PubMed Central

    Dopie, Joseph; Rajakylä, Eeva K.; Joensuu, Merja S.; Huet, Guillaume; Ferrantelli, Evelina; Xie, Tiao; Jäälinoja, Harri; Jokitalo, Eija; Vartiainen, Maria K.

    2015-01-01

    ABSTRACT Nuclear actin plays an important role in many processes that regulate gene expression. Cytoplasmic actin dynamics are tightly controlled by numerous actin-binding proteins, but regulation of nuclear actin has remained unclear. Here, we performed a genome-wide RNA interference (RNAi) screen in Drosophila cells to identify proteins that influence either nuclear polymerization or import of actin. We validate 19 factors as specific hits, and show that Chinmo (known as Bach2 in mammals), SNF4Aγ (Prkag1 in mammals) and Rab18 play a role in nuclear localization of actin in both fly and mammalian cells. We identify several new regulators of cofilin activity, and characterize modulators of both cofilin kinases and phosphatase. For example, Chinmo/Bach2, which regulates nuclear actin levels also in vivo, maintains active cofilin by repressing the expression of the kinase Cdi (Tesk in mammals). Finally, we show that Nup98 and lamin are candidates for regulating nuclear actin polymerization. Our screen therefore reveals new aspects of actin regulation and links nuclear actin to many cellular processes. PMID:26021350

  13. Genome-wide RNAi screen for nuclear actin reveals a network of cofilin regulators.

    PubMed

    Dopie, Joseph; Rajakylä, Eeva K; Joensuu, Merja S; Huet, Guillaume; Ferrantelli, Evelina; Xie, Tiao; Jäälinoja, Harri; Jokitalo, Eija; Vartiainen, Maria K

    2015-07-01

    Nuclear actin plays an important role in many processes that regulate gene expression. Cytoplasmic actin dynamics are tightly controlled by numerous actin-binding proteins, but regulation of nuclear actin has remained unclear. Here, we performed a genome-wide RNA interference (RNAi) screen in Drosophila cells to identify proteins that influence either nuclear polymerization or import of actin. We validate 19 factors as specific hits, and show that Chinmo (known as Bach2 in mammals), SNF4Aγ (Prkag1 in mammals) and Rab18 play a role in nuclear localization of actin in both fly and mammalian cells. We identify several new regulators of cofilin activity, and characterize modulators of both cofilin kinases and phosphatase. For example, Chinmo/Bach2, which regulates nuclear actin levels also in vivo, maintains active cofilin by repressing the expression of the kinase Cdi (Tesk in mammals). Finally, we show that Nup98 and lamin are candidates for regulating nuclear actin polymerization. Our screen therefore reveals new aspects of actin regulation and links nuclear actin to many cellular processes. PMID:26021350

  14. Cofilin-2 controls actin filament length in muscle sarcomeres

    PubMed Central

    Kremneva, Elena; Makkonen, Maarit H.; Skwarek-Maruszewska, Aneta; Gateva, Gergana; Michelot, Alphee; Dominguez, Roberto; Lappalainen, Pekka

    2014-01-01

    SUMMARY ADF/cofilins drive cytoskeletal dynamics by promoting the disassembly of ‘aged’ ADP-actin filaments. Mammals express several ADF/cofilin isoforms, but their specific biochemical activities and cellular functions have not been studied in detail. Here we demonstrate that the muscle-specific isoform cofilin-2 promotes actin filament disassembly in sarcomeres to control the precise length of thin filaments in the contractile apparatus. In contrast to other isoforms, cofilin-2 efficiently binds and disassembles both ADP- and ATP/ADP-Pi-actin filaments. We mapped surface-exposed cofilin-2-specific residues required for ATP-actin binding and propose that these residues function as an ‘actin nucleotide-state sensor’ among ADF/cofilins. The results suggest that cofilin-2 evolved specific biochemical and cellular properties allowing it to control actin dynamics in sarcomeres, where filament pointed ends may contain a mixture of ADP- and ATP/ADP-Pi-actin subunits. Our findings also offer a rationale for why cofilin-2 mutations in humans lead to myopathies. PMID:25373779

  15. Viral Replication Protein Inhibits Cellular Cofilin Actin Depolymerization Factor to Regulate the Actin Network and Promote Viral Replicase Assembly

    PubMed Central

    Kovalev, Nikolay; de Castro Martín, Isabel Fernández; Barajas, Daniel; Risco, Cristina; Nagy, Peter D.

    2016-01-01

    RNA viruses exploit host cells by co-opting host factors and lipids and escaping host antiviral responses. Previous genome-wide screens with Tomato bushy stunt virus (TBSV) in the model host yeast have identified 18 cellular genes that are part of the actin network. In this paper, we show that the p33 viral replication factor interacts with the cellular cofilin (Cof1p), which is an actin depolymerization factor. Using temperature-sensitive (ts) Cof1p or actin (Act1p) mutants at a semi-permissive temperature, we find an increased level of TBSV RNA accumulation in yeast cells and elevated in vitro activity of the tombusvirus replicase. We show that the large p33 containing replication organelle-like structures are located in the close vicinity of actin patches in yeast cells or around actin cable hubs in infected plant cells. Therefore, the actin filaments could be involved in VRC assembly and the formation of large viral replication compartments containing many individual VRCs. Moreover, we show that the actin network affects the recruitment of viral and cellular components, including oxysterol binding proteins and VAP proteins to form membrane contact sites for efficient transfer of sterols to the sites of replication. Altogether, the emerging picture is that TBSV, via direct interaction between the p33 replication protein and Cof1p, controls cofilin activities to obstruct the dynamic actin network that leads to efficient subversion of cellular factors for pro-viral functions. In summary, the discovery that TBSV interacts with cellular cofilin and blocks the severing of existing filaments and the formation of new actin filaments in infected cells opens a new window to unravel the way by which viruses could subvert/co-opt cellular proteins and lipids. By regulating the functions of cofilin and the actin network, which are central nodes in cellular pathways, viruses could gain supremacy in subversion of cellular factors for pro-viral functions. PMID:26863541

  16. Solution Structures and Dynamics of ADF/cofilins UNC-60A and UNC-60B from Caenorhabditis elegans

    PubMed Central

    Shukla, Vaibhav Kumar; Kabra, Ashish; Maheshwari, Diva; Yadav, Rahul; Jain, Anupam; Tripathi, Sarita; Ono, Shoichiro; Kumar, Dinesh; Arora, Ashish

    2015-01-01

    The nematode Caenorhabditis elegans has two ADF/cofilin isoforms, UNC-60A and UNC-60B, which are expressed from the unc60 gene by alternative splicing. UNC-60A has higher activity to cause net depolymerization, and to inhibit polymerization, than UNC-60B. UNC-60B, on the other hand, shows much stronger severing activity than UNC-60A. To understand the structural basis of their functional differences, we have determined the solution structures of UNC-60A and UNC-60B proteins and characterized backbone dynamics. Both UNC-60A and UNC-60B show conserved ADF/cofilin fold. The G-actin binding regions of the two proteins are structurally and dynamically conserved. Accordingly, UNC-60A and UNC-60B individually bind to rabbit muscle ADP-G-actin with high affinities, with Kd of 32.25 nM and 8.62 nM, respectively. The primary differences between these strong and weak severing proteins were observed in the orientation and dynamics of the F-actin binding loop (F-loop). In the strong severing activity isoform UNC-60B, the orientation of F-loop was towards the recently identified F-loop binding region on F-actin, and the F-loop was relatively more flexible with fourteen residues showing motions on nanosecond-picosecond timescale. In contrast, in the weak severing protein isoform UNC-60A, the orientation of F-loop was away from the F-loop binding region and inclined towards its own C-terminal and strand β6. It was also relatively less flexible with only five residues showing motions on nanosecond-picosecond timescale. These differences in structure and dynamics seem to directly correlate to the differential F-actin site-binding and severing properties of UNC-60A and UNC-60B, and other related ADF/cofilin proteins. PMID:25279657

  17. YIH1 is an actin-binding protein that inhibits protein kinase GCN2 and impairs general amino acid control when overexpressed.

    PubMed

    Sattlegger, Evelyn; Swanson, Mark J; Ashcraft, Emily A; Jennings, Jennifer L; Fekete, Richard A; Link, Andrew J; Hinnebusch, Alan G

    2004-07-16

    The general amino acid control (GAAC) enables yeast cells to overcome amino acid deprivation by activation of the alpha subunit of translation initiation factor 2 (eIF2alpha) kinase GCN2 and consequent induction of GCN4, a transcriptional activator of amino acid biosynthetic genes. Binding of GCN2 to GCN1 is required for stimulation of GCN2 kinase activity by uncharged tRNA in starved cells. Here we show that YIH1, when overexpressed, dampens the GAAC response (Gcn- phenotype) by suppressing eIF2alpha phosphorylation by GCN2. The overexpressed YIH1 binds GCN1 and reduces GCN1-GCN2 complex formation, and, consistent with this, the Gcn- phenotype produced by YIH1 overexpression is suppressed by GCN2 overexpression. YIH1 interacts with the same GCN1 fragment that binds GCN2, and this YIH1-GCN1 interaction requires Arg-2259 in GCN1 in vitro and in full-length GCN1 in vivo, as found for GCN2-GCN1 interaction. However, deletion of YIH1 does not increase eIF2alpha phosphorylation or derepress the GAAC, suggesting that YIH1 at native levels is not a general inhibitor of GCN2 activity. We discovered that YIH1 normally resides in a complex with monomeric actin, rather than GCN1, and that a genetic reduction in actin levels decreases the GAAC response. This Gcn- phenotype was partially suppressed by deletion of YIH1, consistent with YIH1-mediated inhibition of GCN2 in actin-deficient cells. We suggest that YIH1 resides in a YIH1-actin complex and may be released for inhibition of GCN2 and stimulation of protein synthesis under specialized conditions or in a restricted cellular compartment in which YIH1 is displaced from monomeric actin. PMID:15126500

  18. Activity-Dependent Dendritic Spine Shrinkage and Growth Involve Downregulation of Cofilin via Distinct Mechanisms

    PubMed Central

    Calabrese, Barbara; Saffin, Jean-Michel; Halpain, Shelley

    2014-01-01

    A current model posits that cofilin-dependent actin severing negatively impacts dendritic spine volume. Studies suggested that increased cofilin activity underlies activity-dependent spine shrinkage, and that reduced cofilin activity induces activity-dependent spine growth. We suggest instead that both types of structural plasticity correlate with decreased cofilin activity. However, the mechanism of inhibition determines the outcome for spine morphology. RNAi in rat hippocampal cultures demonstrates that cofilin is essential for normal spine maintenance. Cofilin-F-actin binding and filament barbed-end production decrease during the early phase of activity-dependent spine shrinkage; cofilin concentration also decreases. Inhibition of the cathepsin B/L family of proteases prevents both cofilin loss and spine shrinkage. Conversely, during activity-dependent spine growth, LIM kinase stimulates cofilin phosphorylation, which activates phospholipase D-1 to promote actin polymerization. These results implicate novel molecular mechanisms and prompt a revision of the current model for how cofilin functions in activity-dependent structural plasticity. PMID:24740405

  19. Cucurbitacin covalent bonding to cysteine thiols: the filamentous-actin severing protein Cofilin1 as an exemplary target

    PubMed Central

    2013-01-01

    Background Cucurbitacins are a class of triterpenoid natural compounds with potent bioactivities that led to their use as traditional remedies, and which continue to attract considerable attention as chemical biology tools and potential therapeutics. One obvious target is the actin-cytoskeleton; treatment with cucurbitacins results in cytoskeletal rearrangements that impact upon motility and cell morphology. Findings Cucurbitacin reacted with protein cysteine thiols as well as dithiothreitol, and we propose that the cucurbitacin mechanism of action is through broad protein thiol modifications that could result in inhibition of numerous protein targets. An example of such a target protein is Cofilin1, whose filamentous actin severing activity is inhibited by cucurbitacin conjugation. Conclusions The implications of these results are that cucurbitacins are unlikely to be improved for selectivity by medicinal chemistry and that their use as chemical biology probes to analyse the role of specific signalling pathways should be undertaken with caution. PMID:23945128

  20. Actin-binding protein regulation by microRNAs as a novel microbial strategy to modulate phagocytosis by host cells: the case of N-Wasp and miR-142-3p

    PubMed Central

    Bettencourt, Paulo; Marion, Sabrina; Pires, David; Santos, Leonor F.; Lastrucci, Claire; Carmo, Nuno; Blake, Jonathon; Benes, Vladimir; Griffiths, Gareth; Neyrolles, Olivier; Lugo-Villarino, Geanncarlo; Anes, Elsa

    2013-01-01

    Mycobacterium tuberculosis (Mtb) is a successful intracellular pathogen that thrives in macrophages (Mφs). There is a need to better understand how Mtb alters cellular processes like phagolysosome biogenesis, a classical determinant of its pathogenesis. A central feature of this bacteria's strategy is the manipulation of Mφ actin. Here, we examined the role of microRNAs (miRNAs) as a potential mechanism in the regulation of actin-mediated events leading to phagocytosis in the context of mycobacteria infection. Given that non-virulent Mycobacterium smegmatis also controls actin filament assembly to prolong its intracellular survival inside host cells, we performed a global transcriptomic analysis to assess the modulation of miRNAs upon M. smegmatis infection of the murine Mφ cell line, J774A.1. This approach identified miR-142-3p as a key candidate to be involved in the regulation of actin dynamics required in phagocytosis. We unequivocally demonstrate that miR-142-3p targets N-Wasp, an actin-binding protein required during microbial challenge. A gain-of-function approach for miR-142-3p revealed a down-regulation of N-Wasp expression accompanied by a decrease of mycobacteria intake, while a loss-of-function approach yielded the reciprocal increase of the phagocytosis process. Equally important, we show Mtb induces the early expression of miR-142-3p and partially down-regulates N-Wasp protein levels in both the murine J774A.1 cell line and primary human Mφs. As proof of principle, the partial siRNA-mediated knock down of N-Wasp resulted in a decrease of Mtb intake by human Mφs, reflected in lower levels of colony-forming units (CFU) counts over time. We therefore propose the modulation of miRNAs as a novel strategy in mycobacterial infection to control factors involved in actin filament assembly and other early events of phagolysosome biogenesis. PMID:23760605

  1. The evolution of the actin binding NET superfamily.

    PubMed

    Hawkins, Timothy J; Deeks, Michael J; Wang, Pengwei; Hussey, Patrick J

    2014-01-01

    The Arabidopsis Networked (NET) superfamily are plant-specific actin binding proteins which specifically label different membrane compartments and identify specialized sites of interaction between actin and membranes unique to plants. There are 13 members of the superfamily in Arabidopsis, which group into four distinct clades or families. NET homologs are absent from the genomes of metazoa and fungi; furthermore, in plantae, NET sequences are also absent from the genome of mosses and more ancient extant plant clades. A single family of the NET proteins is found encoded in the club moss genome, an extant species of the earliest vascular plants. Gymnosperms have examples from families 4 and 3, with a hybrid form of NET1 and 2 which shows characteristics of both NET1 and NET2. In addition to NET3 and 4 families, the NET1 and pollen-expressed NET2 families are found only as independent sequences in Angiosperms. This is consistent with the divergence of reproductive actin. The four families are conserved across Monocots and Eudicots, with the numbers of members of each clade expanding at this point, due, in part, to regions of genome duplication. Since the emergence of the NET superfamily at the dawn of vascular plants, they have continued to develop and diversify in a manner which has mirrored the divergence and increasing complexity of land-plant species. PMID:24926301

  2. The evolution of the actin binding NET superfamily

    PubMed Central

    Hawkins, Timothy J.; Deeks, Michael J.; Wang, Pengwei; Hussey, Patrick J.

    2014-01-01

    The Arabidopsis Networked (NET) superfamily are plant-specific actin binding proteins which specifically label different membrane compartments and identify specialized sites of interaction between actin and membranes unique to plants. There are 13 members of the superfamily in Arabidopsis, which group into four distinct clades or families. NET homologs are absent from the genomes of metazoa and fungi; furthermore, in plantae, NET sequences are also absent from the genome of mosses and more ancient extant plant clades. A single family of the NET proteins is found encoded in the club moss genome, an extant species of the earliest vascular plants. Gymnosperms have examples from families 4 and 3, with a hybrid form of NET1 and 2 which shows characteristics of both NET1 and NET2. In addition to NET3 and 4 families, the NET1 and pollen-expressed NET2 families are found only as independent sequences in Angiosperms. This is consistent with the divergence of reproductive actin. The four families are conserved across Monocots and Eudicots, with the numbers of members of each clade expanding at this point, due, in part, to regions of genome duplication. Since the emergence of the NET superfamily at the dawn of vascular plants, they have continued to develop and diversify in a manner which has mirrored the divergence and increasing complexity of land-plant species. PMID:24926301

  3. Filament assembly by Spire: key residues and concerted actin binding.

    PubMed

    Rasson, Amy S; Bois, Justin S; Pham, Duy Stephen L; Yoo, Haneul; Quinlan, Margot E

    2015-02-27

    The most recently identified class of actin nucleators, WASp homology domain 2 (WH2) nucleators, use tandem repeats of monomeric actin-binding WH2 domains to facilitate actin nucleation. WH2 domains are involved in a wide variety of actin regulatory activities. Structurally, they are expected to clash with interprotomer contacts within the actin filament. Thus, the discovery of their role in nucleation was surprising. Here we use Drosophila Spire (Spir) as a model system to investigate both how tandem WH2 domains can nucleate actin and what differentiates nucleating WH2-containing proteins from their non-nucleating counterparts. We found that the third WH2 domain in Spir (Spir-C or SC) plays a unique role. In the context of a short nucleation construct (containing only two WH2 domains), placement of SC in the N-terminal position was required for the most potent nucleation. We found that the native organization of the WH2 domains with respect to each other is necessary for binding to actin with positive cooperativity. We identified two residues within SC that are critical for its activity. Using this information, we were able to convert a weak synthetic nucleator into one with activity equal to a native Spir construct. Lastly, we found evidence that SC binds actin filaments, in addition to monomers. PMID:25234086

  4. Filament Assembly by Spire: Key Residues and Concerted Actin Binding

    PubMed Central

    Rasson, Amy S.; Bois, Justin S.; Pham, Duy Stephen L.; Yoo, Haneul; Quinlan, Margot E.

    2014-01-01

    The most recently identified class of actin nucleators, WASp Homology domain 2 (WH2) – nucleators, use tandem repeats of monomeric actin-binding WH2 domains to facilitate actin nucleation. WH2 domains are involved in a wide variety of actin regulatory activities. Structurally, they are expected to clash with interprotomer contacts within the actin filament. Thus, the discovery of their role in nucleation was surprising. Here we use Drosophila Spire (Spir) as a model system to investigate both how tandem WH2 domains can nucleate actin and what differentiates nucleating WH2-containing proteins from their non-nucleating counterparts. We found that the third WH2 domain in Spir (Spir-C or Sc), plays a unique role. In the context of a short nucleation construct (containing only two WH2 domains), placement of Sc in the N-terminal position was required for the most potent nucleation. We found that the native organization of the WH2 domains with respect to each other is necessary for binding to actin with positive cooperativity. We identified two residues within Sc that are critical for its activity. Using this information we were able to convert a weak synthetic nucleator into one with activity equal to a native Spir construct. Lastly, we found evidence that Sc binds actin filaments, in addition to monomers. PMID:25234086

  5. Tailor-Made Ezrin Actin Binding Domain to Probe Its Interaction with Actin In-Vitro

    PubMed Central

    Shrivastava, Rohini; Köster, Darius; Kalme, Sheetal; Mayor, Satyajit; Neerathilingam, Muniasamy

    2015-01-01

    Ezrin, a member of the ERM (Ezrin/Radixin/Moesin) protein family, is an Actin-plasma membrane linker protein mediating cellular integrity and function. In-vivo study of such interactions is a complex task due to the presence of a large number of endogenous binding partners for both Ezrin and Actin. Further, C-terminal actin binding capacity of the full length Ezrin is naturally shielded by its N-terminal, and only rendered active in the presence of Phosphatidylinositol bisphosphate (PIP2) or phosphorylation at the C-terminal threonine. Here, we demonstrate a strategy for the design, expression and purification of constructs, combining the Ezrin C-terminal actin binding domain, with functional elements such as fusion tags and fluorescence tags to facilitate purification and fluorescence microscopy based studies. For the first time, internal His tag was employed for purification of Ezrin actin binding domain based on in-silico modeling. The functionality (Ezrin-actin interaction) of these constructs was successfully demonstrated by using Total Internal Reflection Fluorescence Microscopy. This design can be extended to other members of the ERM family as well. PMID:25860910

  6. Gcn1 and actin binding to Yih1: implications for activation of the eIF2 kinase GCN2.

    PubMed

    Sattlegger, Evelyn; Barbosa, João A R G; Moraes, Maria Carolina S; Martins, Rafael M; Hinnebusch, Alan G; Castilho, Beatriz A

    2011-03-25

    Yeast Yih1 protein and its mammalian ortholog IMPACT, abundant in neurons, are inhibitors of Gcn2, a kinase involved in amino acid homeostasis, stress response, and memory formation. Like Gcn2, Yih1/IMPACT harbors an N-terminal RWD domain that mediates binding to the Gcn2 activator Gcn1. Yih1 competes with Gcn2 for Gcn1 binding, thus inhibiting Gcn2. Yih1 also binds G-actin. Here, we show that Yih1-actin interaction is independent of Gcn1 and that Yih1-Gcn1 binding does not require actin. The Yih1 RWD (residues 1-132) was sufficient for Gcn2 inhibition and Gcn1 binding, but not for actin binding, showing that actin binding is dispensable for inhibiting Gcn2. Actin binding required Yih1 residues 68-258, encompassing part of the RWD and the C-terminal "ancient domain"; however, residues Asp-102 and Glu-106 in helix3 of the RWD were essential for Gcn1 binding and Gcn2 inhibition but dispensable for actin binding. Thus, the Gcn1- and actin-binding sites overlap in the RWD but have distinct binding determinants. Unexpectedly, Yih1 segment 68-258 was defective for inhibiting Gcn2 even though it binds Gcn1 at higher levels than does full-length Yih1. This and other results suggest that Yih1 binds with different requirements to distinct populations of Gcn1 molecules, and its ability to disrupt Gcn1-Gcn2 complexes is dependent on a complete RWD and hindered by actin binding. Modeling of the ancient domain on the bacterial protein YigZ showed peculiarities to the eukaryotic and prokaryotic lineages, suggesting binding sites for conserved cellular components. Our results support a role for Yih1 in a cross-talk between the cytoskeleton and translation. PMID:21239490

  7. Identification and characterization of the actin-binding motif of phostensin.

    PubMed

    Wang, Tzu-Fan; Lai, Ning-Sheng; Huang, Kuang-Yung; Huang, Hsien-Lu; Lu, Ming-Chi; Lin, Yu-Shan; Chen, Chun-Yu; Liu, Su-Qin; Lin, Ta-Hsien; Huang, Hsien-Bin

    2012-01-01

    Phostensin, a protein phosphatase 1 F-actin cytoskeleton-targeting subunit encoded by KIAA1949, consists of 165 amino acids and caps the pointed ends of actin filaments. Sequence alignment analyses suggest that the C-terminal region of phostensin, spanning residues 129 to 155, contains a consensus actin-binding motif. Here, we have verified the existence of an actin-binding motif in the C-terminal domain of phostensin using colocalization, F-actin co-sedimentation and single filament binding assays. Our data indicate that the N-terminal region of phostensin (1-129) cannot bind to actin filaments and cannot retard the pointed end elongation of gelsolin-actin seeds. Furthermore, the C-terminal region of phostensin (125-165) multiply bind to the sides of actin filaments and lacks the ability to block the pointed end elongation, suggesting that the actin-binding motif is located in the C-terminal region of the phostensin. Further analyses indicate that phostensin binding to the pointed end of actin filament requires N-terminal residues 35 to 51. These results suggest that phostensin might fold into a rigid structure, allowing the N-terminus to sterically hinder the binding of C-terminus to the sides of actin filament, thus rendering phostensin binding to the pointed ends of actin filaments. PMID:23443105

  8. 25 Years of Tension over Actin Binding to the Cadherin Cell Adhesion Complex: The Devil is in the Details.

    PubMed

    Nelson, W James; Weis, William I

    2016-07-01

    Over the past 25 years, there has been a conceptual (re)evolution in understanding how the cadherin cell adhesion complex, which contains F-actin-binding proteins, binds to the actin cytoskeleton. There is now good synergy between structural, biochemical, and cell biological results that the cadherin-catenin complex binds to F-actin under force. PMID:27166091

  9. High-Resolution Crystal Structures of Villin Headpiece nad Mutants with Reduced F-Actin Binding Activity

    SciTech Connect

    Meng,J.; Vardar, D.; Wang, Y.; Guo, H.; Head, J.; McKnight, C.

    2005-01-01

    Villin-type headpiece domains are approximately 70 amino acid modular motifs found at the C terminus of a variety of actin cytoskeleton-associated proteins. The headpiece domain of villin, a protein found in the actin bundles of the brush border epithelium, is of interest both as a compact F-actin binding domain and as a model folded protein. We have determined the high-resolution crystal structures of chicken villin headpiece (HP67) at 1.4 Angstrom resolution as well as two mutants, R37A and W64Y, at 1.45 and 1.5 Angstrom resolution, respectively. Replacement of R37 causes a 5-fold reduction in F-actin binding affinity in sedimentation assays. Replacement of W64 results in a much more drastic reduction in F-actin binding affinity without significant changes in headpiece structure or stability. The detailed comparison of these crystal structures with each other and to our previously determined NMR structures of HP67 and the 35-residue autonomously folding subdomain in villin headpiece, HP35, provides the details of the headpiece fold and further defines the F-actin binding site of villin-type headpiece domains.

  10. Locomotor proteins in tissues of primary tumors and metastases of ovarian and breast cancer

    NASA Astrophysics Data System (ADS)

    Kondakova, I. V.; Yunusova, N. V.; Spirina, L. V.; Shashova, E. E.; Kolegova, E. S.; Kolomiets, L. A.; Slonimskaya, E. M.; Villert, A. B.

    2016-08-01

    The paper discusses the capability for active movement in an extracellular matrix, wherein remodeling of the cytoskeleton by actin binding proteins plays a significant role in metastases formation. We studied the expression of actin binding proteins and β-catenin in tissues of primary tumors and metastases of ovarian and breast cancer. Contents of p45 Ser β-catenin and the actin severing protein gelsolin were decreased in metastases of ovarian cancer relative to primary tumors. The level of the cofilin, functionally similar to gelsolin, was significantly higher in metastases compared to primary ovarian and breast tumor tissue. In breast cancer, significant increase in the number of an actin monomer binder protein thymosin-β4 was observed in metastases as compared to primary tumors. The data obtained suggest the involvement of locomotor proteins in metastases formation in ovarian and breast cancer.

  11. F-actin binds to the cytoplasmic surface of ponticulin, a 17-kD integral glycoprotein from Dictyostelium discoideum plasma membranes

    PubMed Central

    1987-01-01

    F-actin affinity chromatography and immunological techniques are used to identify actin-binding proteins in purified Dictyostelium discoideum plasma membranes. A 17-kD integral glycoprotein (gp17) consistently elutes from F-actin columns as the major actin-binding protein under a variety of experimental conditions. The actin-binding activity of gp17 is identical to that of intact plasma membranes: it resists extraction with 0.1 N NaOH, 1 mM dithiothreitol (DTT); it is sensitive to ionic conditions; it is stable over a wide range of pH; and it is eliminated by proteolysis, denaturation with heat, or treatment with DTT and N- ethylmaleimide. gp17 may be responsible for much of the actin-binding activity of plasma membranes since monovalent antibody fragments (Fab) directed primarily against gp17 inhibit actin-membrane binding by 96% in sedimentation assays. In contrast, Fab directed against cell surface determinants inhibit binding by only 0-10%. The actin-binding site of gp17 appears to be located on the cytoplasmic surface of the membrane since Fab against this protein continue to inhibit 96% of actin- membrane binding even after extensive adsorption against cell surfaces. gp17 is abundant in the plasma membrane, constituting 0.4-1.0% of the total membrane protein. A transmembrane orientation of gp17 is suggested since, in addition to the cytoplasmic localization of the actin-binding site, extracellular determinants of gp17 are identified. gp17 is surface-labeled by sulfo-N-hydroxy-succinimido-biotin, a reagent that cannot penetrate the cell membrane. Also, gp17 is glycosylated since it is specifically bound by the lectin, concanavalin A. We propose that gp17 is a major actin-binding protein that is important for connecting the plasma membrane to the underlying microfilament network. Therefore, we have named this protein "ponticulin" from the Latin word, ponticulus, which means small bridge. PMID:3312238

  12. How cofilin severs an actin filament.

    PubMed

    De La Cruz, Enrique M

    2009-05-15

    The actin regulatory protein, cofilin, promotes actin assembly dynamics by severing filaments and increasing the number of ends from which subunits add and dissociate. Recent studies provide biophysical descriptions of cooperative filament interactions in energetic, mechanical and structural terms. A one-dimensional Ising model with nearest-neighbor interactions permits thermodynamic analysis of cooperative binding and indicates that one or a few cofilin molecules can sever a filament. Binding and cooperative interactions are entropically driven. A significant fraction of the binding free energy results from the linked dissociation of filament-associated ions (polyelectrolyte effect), which modulate filament structure, stability and mechanics. The remaining binding free energy and essentially all of the cooperative free energy arise from the enhanced conformational dynamics of the cofilactin complex. Filament mechanics are modulated by cofilin such that cofilin-saturated filaments are approximately 10- to 20-fold more compliant in bending and twisting than bare filaments. Cofilin activity is well described by models in which discontinuities in topology, mechanics and conformational dynamics generate stress concentration and promote fracture at junctions of bare and decorated segments, analogous to the grain boundary fracture of crystalline materials and the thermally driven formation of shear transformation zones in colloidal glass. PMID:20700473

  13. Concentration profiles of actin-binding molecules in lamellipodia

    NASA Astrophysics Data System (ADS)

    Falcke, Martin

    2016-04-01

    Motile cells form lamellipodia in the direction of motion, which are flat membrane protrusions containing an actin filament network. The network flows rearward relative to the leading edge of the lamellipodium due to actin polymerization at the front. Thus, actin binding molecules are subject to transport towards the rear of the cell in the bound state and diffuse freely in the unbound state. We analyze this reaction-diffusion-advection process with respect to the concentration profiles of these species and provide an analytic approximation for them. Network flow may cause a depletion zone of actin binding molecules close to the leading edge. The existence of such zone depends on the free molecule concentration in the cell body, on the ratio of the diffusion length to the distance bound molecules travel rearward with the flow before dissociating, and the ratio of the diffusion length to the width of the region with network flow and actin binding. Our calculations suggest the existence of depletion zones for the F-actin cross-linkers filamin and α-actinin in fish keratocytes (and other cell types), which is in line with the small elastic moduli of the F-actin network close to the leading edge found in measurements of the force motile cells are able to exert.

  14. A human β-III-spectrin spinocerebellar ataxia type 5 mutation causes high-affinity F-actin binding

    PubMed Central

    Avery, Adam W.; Crain, Jonathan; Thomas, David D.; Hays, Thomas S.

    2016-01-01

    Spinocerebellar ataxia type 5 (SCA5) is a human neurodegenerative disease that stems from mutations in the SPTBN2 gene encoding the protein β-III-spectrin. Here we investigated the molecular consequence of a SCA5 missense mutation that results in a L253P substitution in the actin-binding domain (ABD) of β-III-spectrin. We report that the L253P substitution in the isolated β-III-spectrin ABD causes strikingly high F-actin binding affinity (Kd = 75.5 nM) compared to the weak F-actin binding affinity of the wild-type ABD (Kd = 75.8 μM). The mutation also causes decreased thermal stability (Tm = 44.6 °C vs 59.5 °C). Structural analyses indicate that leucine 253 is in a loop at the interface of the tandem calponin homology (CH) domains comprising the ABD. Leucine 253 is predicted to form hydrophobic contacts that bridge the CH domains. The decreased stability of the mutant indicates that these bridging interactions are probably disrupted, suggesting that the high F-actin binding affinity of the mutant is due to opening of the CH domain interface. These results support a fundamental role for leucine 253 in regulating opening of the CH domain interface and binding of the ABD to F-actin. This study indicates that high-affinity actin binding of L253P β-III-spectrin is a likely driver of neurodegeneration. PMID:26883385

  15. A human β-III-spectrin spinocerebellar ataxia type 5 mutation causes high-affinity F-actin binding.

    PubMed

    Avery, Adam W; Crain, Jonathan; Thomas, David D; Hays, Thomas S

    2016-01-01

    Spinocerebellar ataxia type 5 (SCA5) is a human neurodegenerative disease that stems from mutations in the SPTBN2 gene encoding the protein β-III-spectrin. Here we investigated the molecular consequence of a SCA5 missense mutation that results in a L253P substitution in the actin-binding domain (ABD) of β-III-spectrin. We report that the L253P substitution in the isolated β-III-spectrin ABD causes strikingly high F-actin binding affinity (Kd = 75.5 nM) compared to the weak F-actin binding affinity of the wild-type ABD (Kd = 75.8 μM). The mutation also causes decreased thermal stability (Tm = 44.6 °C vs 59.5 °C). Structural analyses indicate that leucine 253 is in a loop at the interface of the tandem calponin homology (CH) domains comprising the ABD. Leucine 253 is predicted to form hydrophobic contacts that bridge the CH domains. The decreased stability of the mutant indicates that these bridging interactions are probably disrupted, suggesting that the high F-actin binding affinity of the mutant is due to opening of the CH domain interface. These results support a fundamental role for leucine 253 in regulating opening of the CH domain interface and binding of the ABD to F-actin. This study indicates that high-affinity actin binding of L253P β-III-spectrin is a likely driver of neurodegeneration. PMID:26883385

  16. Three-Dimensional Structure of Cofilin Bound to Monomeric Actin Derived by Structural Mass Spectrometry Data

    SciTech Connect

    Amisha Kamal,J.; Benchaar, S.; Takamoto, K.; Reisler, E.; Chance, M.

    2007-01-01

    The cytoskeletal protein, actin, has its structure and function regulated by cofilin. In the absence of an atomic resolution structure for the actin/cofilin complex, the mechanism of cofilin regulation is poorly understood. Theoretical studies based on the similarities of cofilin and gelsolin segment 1 proposed the cleft between subdomains 1 and 3 in actin as the cofilin binding site. We used radiolytic protein footprinting with mass spectrometry and molecular modeling to provide an atomic model of how cofilin binds to monomeric actin. Footprinting data suggest that cofilin binds to the cleft between subdomains 1 and 2 in actin and that cofilin induces further closure of the actin nucleotide cleft. Site-specific fluorescence data confirm these results. The model identifies key ionic and hydrophobic interactions at the binding interface, including hydrogen-bonding between His-87 of actin to Ser-89 of cofilin that may control the charge dependence of cofilin binding. This model and its implications fill an especially important niche in the actin field, owing to the fact that ongoing crystallization efforts of the actin/cofilin complex have so far failed. This 3D binary complex structure is derived from a combination of solution footprinting data and computational approaches and outlines a general method for determining the structure of such complexes.

  17. Cofilin Inhibition Restores Neuronal Cell Death in Oxygen-Glucose Deprivation Model of Ischemia.

    PubMed

    Madineni, Anusha; Alhadidi, Qasim; Shah, Zahoor A

    2016-03-01

    Ischemia is a condition associated with decreased blood supply to the brain, eventually leading to death of neurons. It is associated with a diverse cascade of responses involving both degenerative and regenerative mechanisms. At the cellular level, the changes are initiated prominently in the neuronal cytoskeleton. Cofilin, a cytoskeletal actin severing protein, is known to be involved in the early stages of apoptotic cell death. Evidence supports its intervention in the progression of disease states like Alzheimer's and ischemic kidney disease. In the present study, we have hypothesized the possible involvement of cofilin in ischemia. Using PC12 cells and mouse primary cultures of cortical neurons, we investigated the potential role of cofilin in ischemia in two different in vitro ischemic models: chemical induced oxidative stress and oxygen-glucose deprivation/reperfusion (OGD/R). The expression profile studies demonstrated a decrease in phosphocofilin levels in all models of ischemia, implying stress-induced cofilin activation. Furthermore, calcineurin and slingshot 1L (SSH) phosphatases were found to be the signaling mediators of the cofilin activation. In primary cultures of cortical neurons, cofilin was found to be significantly activated after 1 h of OGD. To delineate the role of activated cofilin in ischemia, we knocked down cofilin by small interfering RNA (siRNA) technique and tested the impact of cofilin silencing on neuronal viability. Cofilin siRNA-treated neurons showed a significant reduction of cofilin levels in all treatment groups (control, OGD, and OGD/R). Additionally, cofilin siRNA-reduced cofilin mitochondrial translocation and caspase 3 cleavage, with a concomitant increase in neuronal viability. These results strongly support the active role of cofilin in ischemia-induced neuronal degeneration and apoptosis. We believe that targeting this protein mediator has a potential for therapeutic intervention in ischemic brain injury and stroke

  18. Pathogenic microbes manipulate cofilin activity to subvert actin cytoskeleton.

    PubMed

    Zheng, Kai; Kitazato, Kaio; Wang, Yifei; He, Zhendan

    2016-09-01

    Actin-depolymerizing factor (ADF)/cofilin proteins are key players in controlling the temporal and spatial extent of actin dynamics, which is crucial for mediating host-pathogen interactions. Pathogenic microbes have evolved molecular mechanisms to manipulate cofilin activity to subvert the actin cytoskeletal system in host cells, promoting their internalization into the target cells, modifying the replication niche and facilitating their intracellular and intercellular dissemination. The study of how these pathogens exploit cofilin pathways is crucial for understanding infectious disease and providing potential targets for drug therapies. PMID:25853495

  19. Rabies virus inactivates cofilin to facilitate viral budding and release.

    PubMed

    Zan, Jie; An, Shu-Ting; Mo, Kai-Kun; Zhou, Jian-Wei; Liu, Juan; Wang, Hai-Long; Yan, Yan; Liao, Min; Zhou, Ji-Yong

    2016-09-01

    Cytoplasmic actin and actin-associated proteins have been identified in RABV particles. Although actin is involved in RABV entry into cells, the specific role of actin in RABV budding and release remains unknown. Our study found that RABV M protein-mediated virion budding depends on intact actin filaments. Confocal microscopy demonstrated a block to virions budding, with a number of M protein-mediated budding vesicles detained in the cell cytoplasm. Furthermore, RABV infection resulted in inactivation of cofilin and upregulation of phosphorylated cofilin. Knockdown of cofilin reduced RABV release. These results for the first time indicate that RABV infection resulted in upregulation of phosphorylated cofilin to facililtate actin polymerization for virus budding. PMID:27396619

  20. The actin binding site of thymosin beta 4 mapped by mutational analysis.

    PubMed Central

    Van Troys, M; Dewitte, D; Goethals, M; Carlier, M F; Vandekerckhove, J; Ampe, C

    1996-01-01

    We characterized in detail the actin binding site of the small actin-sequestering protein thymosin beta 4 (T beta 4) using chemically synthesized full-length T beta 4 variants. The N-terminal part (residues 1-16) and a hexapeptide motif (residues 17-22) form separate structural entities. In both, we identified charged and hydrophobic residues that participate in the actin interaction using chemical cross-linking, complex formation in native gels and actin-sequestering experiments. Quantitative data on the activity of the variants and circular dichroism experiments allow to present a model in which the N-terminal part needs to adopt an alpha-helix for actin binding and interacts through a patch of hydrophobic residues (6M-I-F12) on one side of this helix. Also, electrostatic contacts between actin and lysine residues 18, in the motif, and 14, in the N-terminal alpha-helix, appear important for binding. The residues critical for contacting actin are conserved throughout the beta-thymosin family and in addition to this we identify a similar pattern in the C-terminal headpiece of villin and dematin. Images PMID:8617195

  1. Human RNASET2 derivatives as potential anti-angiogenic agents: actin binding sequence identification and characterization

    PubMed Central

    Nesiel-Nuttman, Liron; Doron, Shani; Schwartz, Betty; Shoseyov, Oded

    2015-01-01

    Human RNASET2 (hRNASET2) has been demonstrated to exert antiangiogenic and antitumorigenic effects independent of its ribonuclease capacity. We suggested that RNASET2 exerts its antiangiogenic and antitumorigenic activities via binding to actin and consequently inhibits cell motility. We focused herein on the identification of the actin binding site of hRNASET2 using defined sequences encountered within the whole hRNASET2 protein. For that purpose we designed 29 different hRNASET2-derived peptides. The 29 peptides were examined for their ability to bind immobilized actin. Two selected peptides-A103-Q159 consisting of 57 amino acids and peptide K108-K133 consisting of 26 amino acids were demonstrated to have the highest actin binding ability and concomitantly the most potent anti-angiogenic activity. Further analyses on the putative mechanisms associated with angiogenesis inhibition exerted by peptide K108-K133 involved its location during treatment within the HUVE cells. Peptide K108-K133 readily penetrates the cell membrane within 10 min of incubation. In addition, supplementation with angiogenin delays the entrance of peptide K108-K133 to the cell suggesting competition on the same cell internalization route. The peptide was demonstrated to co-localize with angiogenin, suggesting that both molecules bind analogous cellular epitopes, similar to our previously reported data for ACTIBIND and trT2-50. PMID:25815360

  2. Human RNASET2 derivatives as potential anti-angiogenic agents: actin binding sequence identification and characterization.

    PubMed

    Nesiel-Nuttman, Liron; Doron, Shani; Schwartz, Betty; Shoseyov, Oded

    2015-01-01

    Human RNASET2 (hRNASET2) has been demonstrated to exert antiangiogenic and antitumorigenic effects independent of its ribonuclease capacity. We suggested that RNASET2 exerts its antiangiogenic and antitumorigenic activities via binding to actin and consequently inhibits cell motility. We focused herein on the identification of the actin binding site of hRNASET2 using defined sequences encountered within the whole hRNASET2 protein. For that purpose we designed 29 different hRNASET2-derived peptides. The 29 peptides were examined for their ability to bind immobilized actin. Two selected peptides-A103-Q159 consisting of 57 amino acids and peptide K108-K133 consisting of 26 amino acids were demonstrated to have the highest actin binding ability and concomitantly the most potent anti-angiogenic activity. Further analyses on the putative mechanisms associated with angiogenesis inhibition exerted by peptide K108-K133 involved its location during treatment within the HUVE cells. Peptide K108-K133 readily penetrates the cell membrane within 10 min of incubation. In addition, supplementation with angiogenin delays the entrance of peptide K108-K133 to the cell suggesting competition on the same cell internalization route. The peptide was demonstrated to co-localize with angiogenin, suggesting that both molecules bind analogous cellular epitopes, similar to our previously reported data for ACTIBIND and trT2-50. PMID:25815360

  3. Assessments of urine cofilin-1 in patients hospitalized in the intensive care units with acute kidney injury

    NASA Astrophysics Data System (ADS)

    Lee, Yi-Jang; Chao, Cheng-Han; Chang, Ying-Feng; Chou, Chien

    2013-02-01

    The actin depolymerizing factor (ADF)/cofilin protein family has been reported to be associated with ischemia induced renal disorders. Here we examine if cofilin-1 is associated with acute kidney injury (AKI). We exploited a 96-well based fiber-optic biosensor that uses conjugated gold nanoparticles and a sandwich immunoassay to detect the urine cofilin-1 level of AKI patients. The mean urine cofilin-1 level of the AKI patients was two-fold higher than that of healthy adults. The receiver operating characteristic (ROC) curve showed that cofilin-1 is a potential biomarker for discriminating AKI patients from healthy adults for intensive care patients.

  4. F-actin binds to the cytoplasmic surface of ponticulin, a 17-kD integral glycoprotein from Dictyostelium discoideum plasma membranes.

    PubMed

    Wuestehube, L J; Luna, E J

    1987-10-01

    F-actin affinity chromatography and immunological techniques are used to identify actin-binding proteins in purified Dictyostelium discoideum plasma membranes. A 17-kD integral glycoprotein (gp17) consistently elutes from F-actin columns as the major actin-binding protein under a variety of experimental conditions. The actin-binding activity of gp17 is identical to that of intact plasma membranes: it resists extraction with 0.1 N NaOH, 1 mM dithiothreitol (DTT); it is sensitive to ionic conditions; it is stable over a wide range of pH; and it is eliminated by proteolysis, denaturation with heat, or treatment with DTT and N-ethylmaleimide. gp17 may be responsible for much of the actin-binding activity of plasma membranes since monovalent antibody fragments (Fab) directed primarily against gp17 inhibit actin-membrane binding by 96% in sedimentation assays. In contrast, Fab directed against cell surface determinants inhibit binding by only 0-10%. The actin-binding site of gp17 appears to be located on the cytoplasmic surface of the membrane since Fab against this protein continue to inhibit 96% of actin-membrane binding even after extensive adsorption against cell surfaces. gp17 is abundant in the plasma membrane, constituting 0.4-1.0% of the total membrane protein. A transmembrane orientation of gp17 is suggested since, in addition to the cytoplasmic localization of the actin-binding site, extracellular determinants of gp17 are identified. gp17 is surface-labeled by sulfo-N-hydroxy-succinimido-biotin, a reagent that cannot penetrate the cell membrane. Also, gp17 is glycosylated since it is specifically bound by the lectin, concanavalin A. We propose that gp17 is a major actin-binding protein that is important for connecting the plasma membrane to the underlying microfilament network. Therefore, we have named this protein "ponticulin" from the Latin word, ponticulus, which means small bridge. PMID:3312238

  5. State-dependent diffusion of actin-depolymerizing factor/cofilin underlies the enlargement and shrinkage of dendritic spines

    PubMed Central

    Noguchi, Jun; Hayama, Tatsuya; Watanabe, Satoshi; Ucar, Hasan; Yagishita, Sho; Takahashi, Noriko; Kasai, Haruo

    2016-01-01

    Dendritic spines are the postsynaptic sites of most excitatory synapses in the brain, and spine enlargement and shrinkage give rise to long-term potentiation and depression of synapses, respectively. Because spine structural plasticity is accompanied by remodeling of actin scaffolds, we hypothesized that the filamentous actin regulatory protein cofilin plays a crucial role in this process. Here we investigated the diffusional properties of cofilin, the actin-severing and depolymerizing actions of which are activated by dephosphorylation. Cofilin diffusion was measured using fluorescently labeled cofilin fusion proteins and two-photon imaging. We show that cofilins are highly diffusible along dendrites in the resting state. However, during spine enlargement, wild-type cofilin and a phosphomimetic cofilin mutant remain confined to the stimulated spine, whereas a nonphosphorylatable mutant does not. Moreover, inhibition of cofilin phosphorylation with a competitive peptide disables spine enlargement, suggesting that phosphorylated-cofilin accumulation is a key regulator of enlargement, which is localized to individual spines. Conversely, spine shrinkage spreads to neighboring spines, even though triggered by weaker stimuli than enlargement. Diffusion of exogenous cofilin injected into a pyramidal neuron soma causes spine shrinkage and reduced PSD95 in spines, suggesting that diffusion of dephosphorylated endogenous cofilin underlies the spreading of spine shrinkage and long-term depression. PMID:27595610

  6. State-dependent diffusion of actin-depolymerizing factor/cofilin underlies the enlargement and shrinkage of dendritic spines.

    PubMed

    Noguchi, Jun; Hayama, Tatsuya; Watanabe, Satoshi; Ucar, Hasan; Yagishita, Sho; Takahashi, Noriko; Kasai, Haruo

    2016-01-01

    Dendritic spines are the postsynaptic sites of most excitatory synapses in the brain, and spine enlargement and shrinkage give rise to long-term potentiation and depression of synapses, respectively. Because spine structural plasticity is accompanied by remodeling of actin scaffolds, we hypothesized that the filamentous actin regulatory protein cofilin plays a crucial role in this process. Here we investigated the diffusional properties of cofilin, the actin-severing and depolymerizing actions of which are activated by dephosphorylation. Cofilin diffusion was measured using fluorescently labeled cofilin fusion proteins and two-photon imaging. We show that cofilins are highly diffusible along dendrites in the resting state. However, during spine enlargement, wild-type cofilin and a phosphomimetic cofilin mutant remain confined to the stimulated spine, whereas a nonphosphorylatable mutant does not. Moreover, inhibition of cofilin phosphorylation with a competitive peptide disables spine enlargement, suggesting that phosphorylated-cofilin accumulation is a key regulator of enlargement, which is localized to individual spines. Conversely, spine shrinkage spreads to neighboring spines, even though triggered by weaker stimuli than enlargement. Diffusion of exogenous cofilin injected into a pyramidal neuron soma causes spine shrinkage and reduced PSD95 in spines, suggesting that diffusion of dephosphorylated endogenous cofilin underlies the spreading of spine shrinkage and long-term depression. PMID:27595610

  7. Reconstitution of Actin-based Motility by Vasodilator-stimulated Phosphoprotein (VASP) Depends on the Recruitment of F-actin Seeds from the Solution Produced by Cofilin*

    PubMed Central

    Siton, Orit; Bernheim-Groswasser, Anne

    2014-01-01

    Vasodilator-stimulated phosphoprotein (VASP) is active in many filopodium-based and cytoskeleton reorganization processes. It is not fully understood how VASP directly functions in actin-based motility and how regulatory proteins affect its function. Here, we combine bead motility assay and single filament experiments. In the presence of a bundling component, actin bundles that grow from the surface of WT-VASP-coated beads induced movement of the beads. VASP promotes actin-based movement alone, in the absence of other actin nucleators. We propose that at physiological salt conditions VASP nucleation activity is too weak to promote motility and bundle formation. Rather, VASP recruits F-actin seeds from the solution and promotes their elongation. Cofilin has a crucial role in the nucleation of these F-actin seeds, notably under conditions of unfavorable spontaneous actin nucleation. We explored the role of multiple VASP variants. We found that the VASP-F-actin binding domain is required for the recruitment of F-actin seeds from the solution. We also found that the interaction of profilin-actin complexes with the VASP-proline-rich domain and the binding of the VASP-F-actin binding domain to the side of growing filaments is critical for transforming actin polymerization into motion. At the single filament level, profilin mediates both filament elongation rate and VASP anti-capping activity. Binding of profilin-actin complexes increases the polymerization efficiency by VASP but decreases its efficiency as an anti-capper; binding of free profilin creates the opposite effect. Finally, we found that an additional component such as methylcellulose or fascin is required for actin bundle formation and motility mediated by VASP. PMID:25246528

  8. Identification of sucrose synthase as an actin-binding protein

    NASA Technical Reports Server (NTRS)

    Winter, H.; Huber, J. L.; Huber, S. C.; Davies, E. (Principal Investigator)

    1998-01-01

    Several lines of evidence indicate that sucrose synthase (SuSy) binds both G- and F-actin: (i) presence of SuSy in the Triton X-100-insoluble fraction of microsomal membranes (i.e. crude cytoskeleton fraction); (ii) co-immunoprecipitation of actin with anti-SuSy monoclonal antibodies; (iii) association of SuSy with in situ phalloidin-stabilized F-actin filaments; and (iv) direct binding to F-actin, polymerized in vitro. Aldolase, well known to interact with F-actin, interfered with binding of SuSy, suggesting that a common or overlapping binding site may be involved. We postulate that some of the soluble SuSy in the cytosol may be associated with the actin cytoskeleton in vivo.

  9. Site-specific cation release drives actin filament severing by vertebrate cofilin

    PubMed Central

    Kang, Hyeran; Bradley, Michael J.; Cao, Wenxiang; Zhou, Kaifeng; Grintsevich, Elena E.; Michelot, Alphée; Sindelar, Charles V.; Hochstrasser, Mark; De La Cruz, Enrique M.

    2014-01-01

    Actin polymerization powers the directed motility of eukaryotic cells. Sustained motility requires rapid filament turnover and subunit recycling. The essential regulatory protein cofilin accelerates network remodeling by severing actin filaments and increasing the concentration of ends available for elongation and subunit exchange. Although cofilin effects on actin filament assembly dynamics have been extensively studied, the molecular mechanism of cofilin-induced filament severing is not understood. Here we demonstrate that actin filament severing by vertebrate cofilin is driven by the linked dissociation of a single cation that controls filament structure and mechanical properties. Vertebrate cofilin only weakly severs Saccharomyces cerevisiae actin filaments lacking this “stiffness cation” unless a stiffness cation-binding site is engineered into the actin molecule. Moreover, vertebrate cofilin rescues the viability of a S. cerevisiae cofilin deletion mutant only when the stiffness cation site is simultaneously introduced into actin, demonstrating that filament severing is the essential function of cofilin in cells. This work reveals that site-specific interactions with cations serve a key regulatory function in actin filament fragmentation and dynamics. PMID:25468977

  10. Hypertrophic cardiomyopathy mutations in the calponin-homology domain of ACTN2 affect actin binding and cardiomyocyte Z-disc incorporation

    PubMed Central

    Haywood, Natalie J.; Wolny, Marcin; Rogers, Brendan; Trinh, Chi H.; Shuping, Yu; Edwards, Thomas A.; Peckham, Michelle

    2016-01-01

    α-Actinin-2 (ACTN2) is the only muscle isoform of α-actinin expressed in cardiac muscle. Mutations in this protein have been implicated in mild to moderate forms of hypertrophic cardiomyopathy (HCM). We have investigated the effects of two mutations identified from HCM patients, A119T and G111V, on the secondary and tertiary structure of a purified actin binding domain (ABD) of ACTN2 by circular dichroism and X-ray crystallography, and show small but distinct changes for both mutations. We also find that both mutants have reduced F-actin binding affinity, although the differences are not significant. The full length mEos2 tagged protein expressed in adult cardiomyocytes shows that both mutations additionally affect Z-disc localization and dynamic behaviour. Overall, these two mutations have small effects on structure, function and behaviour, which may contribute to a mild phenotype for this disease. PMID:27287556

  11. Actin binding and proline rich motifs of CR16 play redundant role in growth of vrp1Delta cells.

    PubMed

    Meng, Lei; Rajmohan, Rajamuthiah; Yu, Shangjuan; Thanabalu, Thirumaran

    2007-05-25

    CR16, (Glucocorticoid-regulated) belongs to the verprolin family of proteins which are characterized by the presence of a V domain (verprolin) at the N-terminal. Expression of CR16 suppressed the growth and endocytosis defect of vrp1Delta strain without correcting the actin patch polarization defect. The V domain of CR16 is critical for suppression of the growth defect of vrp1Delta strain but not for localisation to cortical actin patches. Mutations in the actin binding motif alone did not abolish the activity of CR16 but the mutations in combination with deletion of N-terminal proline rich motif abolished the ability of CR16 to suppress the growth defect. This suggests that the V domain of CR16 has two functionally redundant motifs and either one of these motifs is sufficient for suppressing the growth defect of vrp1Delta strain. This is in contrast to the observation that both WIP and WIRE require the actin binding motif for their activity. PMID:17418095

  12. Determination of urine cofilin-1 level in acute kidney injury using a high-throughput localized surface plasmon-coupled fluorescence biosensor

    NASA Astrophysics Data System (ADS)

    Chang, Ying-Feng; Chao, Cheng-Han; Lin, Lih-Yuan; Tsai, Cheng-Han; Chou, Chien; Lee, Yi-Jang

    2014-01-01

    The actin-depolymerizing factor (ADF)/cofilin protein family has been reported to be associated with ischemia-induced renal disorders. We examine whether cofilin-1 is associated with acute kidney injury (AKI) using human urine samples. We exploited a 96-well based high-throughput biosensor that uses gold nanoparticles and a sandwich immunoassay to detect the urine cofilin-1 level of AKI patients. The mean urine cofilin-1 level of the AKI patients (n=37 from 47 cases analyzed) was twofold higher than that of healthy adults (n=21 from 29 cases analyzed). The receiver operating characteristic (ROC) curve showed that cofilin-1 was acceptable for discriminating AKI patients from healthy adults. However, an increase of the sample size is required to conclude the importance of urine cofilin-1 on AKI diagnosis, and the high-throughput ultrasensitive biosensor used in this study would greatly accelerate the measurement of urine cofilin-1 in an increased sample size.

  13. Activation of F-Actin Binding Capacity of Ezrin: Synergism of PIP2 Interaction and Phosphorylation

    PubMed Central

    Bosk, Sabine; Braunger, Julia A.; Gerke, Volker; Steinem, Claudia

    2011-01-01

    Ezrin is a membrane-cytoskeleton linker protein that can bind F-actin in its active conformation. Several means of regulation of ezrin's activity have been described including phosphorylation of Thr-567 and binding of L-α-phosphatidylinositol-4,5-bisphosphate (PIP2). However, the relative contributions of these events toward activation of the protein and their potential interdependence are not known. We developed an assay based on solid-supported membranes, to which different ezrin mutants (ezrin T567A (inactive mutant), wild-type, and T567D (active pseudophosphorylated mutant)) were bound, that enabled us to analyze the influence of phosphorylation and PIP2 binding on ezrin's activation state in vitro. The lipid bilayers employed contained either DOGS-NTA-Ni to bind the proteins via an N-terminal His-tag, or PIP2, to which ezrin binds via specific binding sites located in the N-terminal region of the protein. Quantitative analysis of the binding behavior of all three proteins to the two different receptor lipids revealed that all three bind with high affinity and specificity to the two receptor lipids. Fluorescence microscopy on ezrin-decorated solid-supported membranes showed that, dependent on the mode of binding and the phosphorylation state, ezrin is capable of binding actin filaments. A clear synergism between phosphorylation and the receptor lipid PIP2 was observed, suggesting a conformational switch from the dormant to the active, F-actin binding state by recognition of PIP2, which is enhanced by the phosphorylation. PMID:21463584

  14. A single charge in the actin binding domain of fascin can independently tune the linear and non-linear response of an actin bundle network.

    PubMed

    Maier, M; Müller, K W; Heussinger, C; Köhler, S; Wall, W A; Bausch, A R; Lieleg, O

    2015-05-01

    Actin binding proteins (ABPs) not only set the structure of actin filament assemblies but also mediate the frequency-dependent viscoelastic moduli of cross-linked and bundled actin networks. Point mutations in the actin binding domain of those ABPs can tune the association and dissociation dynamics of the actin/ABP bond and thus modulate the network mechanics both in the linear and non-linear response regime. We here demonstrate how the exchange of a single charged amino acid in the actin binding domain of the ABP fascin triggers such a modulation of the network rheology. Whereas the overall structure of the bundle networks is conserved, the transition point from strain-hardening to strain-weakening sensitively depends on the cross-linker off-rate and the applied shear rate. Our experimental results are consistent both with numerical simulations of a cross-linked bundle network and a theoretical description of the bundle network mechanics which is based on non-affine bending deformations and force-dependent cross-link dynamics. PMID:26004635

  15. The Actin Binding Domain of βI-Spectrin Regulates the Morphological and Functional Dynamics of Dendritic Spines

    PubMed Central

    Nestor, Michael W.; Cai, Xiang; Stone, Michele R.; Bloch, Robert J.; Thompson, Scott M.

    2011-01-01

    Actin microfilaments regulate the size, shape and mobility of dendritic spines and are in turn regulated by actin binding proteins and small GTPases. The βI isoform of spectrin, a protein that links the actin cytoskeleton to membrane proteins, is present in spines. To understand its function, we expressed its actin-binding domain (ABD) in CA1 pyramidal neurons in hippocampal slice cultures. The ABD of βI-spectrin bundled actin in principal dendrites and was concentrated in dendritic spines, where it significantly increased the size of the spine head. These effects were not observed after expression of homologous ABDs of utrophin, dystrophin, and α-actinin. Treatment of slice cultures with latrunculin-B significantly decreased spine head size and decreased actin-GFP fluorescence in cells expressing the ABD of α-actinin, but not the ABD of βI-spectrin, suggesting that its presence inhibits actin depolymerization. We also observed an increase in the area of GFP-tagged PSD-95 in the spine head and an increase in the amplitude of mEPSCs at spines expressing the ABD of βI-spectrin. The effects of the βI-spectrin ABD on spine size and mEPSC amplitude were mimicked by expressing wild-type Rac3, a small GTPase that co-immunoprecipitates specifically with βI-spectrin in extracts of cultured cortical neurons. Spine size was normal in cells co-expressing a dominant negative Rac3 construct with the βI-spectrin ABD. We suggest that βI-spectrin is a synaptic protein that can modulate both the morphological and functional dynamics of dendritic spines, perhaps via interaction with actin and Rac3. PMID:21297961

  16. Mutations in the N-terminal actin-binding domain of filamin C cause a distal myopathy.

    PubMed

    Duff, Rachael M; Tay, Valerie; Hackman, Peter; Ravenscroft, Gianina; McLean, Catriona; Kennedy, Paul; Steinbach, Alina; Schöffler, Wiebke; van der Ven, Peter F M; Fürst, Dieter O; Song, Jaeguen; Djinović-Carugo, Kristina; Penttilä, Sini; Raheem, Olayinka; Reardon, Katrina; Malandrini, Alessandro; Gambelli, Simona; Villanova, Marcello; Nowak, Kristen J; Williams, David R; Landers, John E; Brown, Robert H; Udd, Bjarne; Laing, Nigel G

    2011-06-10

    Linkage analysis of the dominant distal myopathy we previously identified in a large Australian family demonstrated one significant linkage region located on chromosome 7 and encompassing 18.6 Mbp and 151 genes. The strongest candidate gene was FLNC because filamin C, the encoded protein, is muscle-specific and associated with myofibrillar myopathy. Sequencing of FLNC cDNA identified a c.752T>C (p.Met251Thr) mutation in the N-terminal actin-binding domain (ABD); this mutation segregated with the disease and was absent in 200 controls. We identified an Italian family with the same phenotype and found a c.577G>A (p.Ala193Thr) filamin C ABD mutation that segregated with the disease. Filamin C ABD mutations have not been described, although filamin A and filamin B ABD mutations cause multiple musculoskeletal disorders. The distal myopathy phenotype and muscle pathology in the two families differ from myofibrillar myopathies caused by filamin C rod and dimerization domain mutations because of the distinct involvement of hand muscles and lack of pathological protein aggregation. Thus, like the position of FLNA and B mutations, the position of the FLNC mutation determines disease phenotype. The two filamin C ABD mutations increase actin-binding affinity in a manner similar to filamin A and filamin B ABD mutations. Cell-culture expression of the c.752T>C (p.Met251)Thr mutant filamin C ABD demonstrated reduced nuclear localization as did mutant filamin A and filamin B ABDs. Expression of both filamin C ABD mutants as full-length proteins induced increased aggregation of filamin. We conclude filamin C ABD mutations cause a recognizable distal myopathy, most likely through increased actin affinity, similar to the pathological mechanism of filamin A and filamin B ABD mutations. PMID:21620354

  17. Mutations in the N-terminal Actin-Binding Domain of Filamin C Cause a Distal Myopathy

    PubMed Central

    Duff, Rachael M.; Tay, Valerie; Hackman, Peter; Ravenscroft, Gianina; McLean, Catriona; Kennedy, Paul; Steinbach, Alina; Schöffler, Wiebke; van der Ven, Peter F.M.; Fürst, Dieter O.; Song, Jaeguen; Djinović-Carugo, Kristina; Penttilä, Sini; Raheem, Olayinka; Reardon, Katrina; Malandrini, Alessandro; Gambelli, Simona; Villanova, Marcello; Nowak, Kristen J.; Williams, David R.; Landers, John E.; Brown, Robert H.; Udd, Bjarne; Laing, Nigel G.

    2011-01-01

    Linkage analysis of the dominant distal myopathy we previously identified in a large Australian family demonstrated one significant linkage region located on chromosome 7 and encompassing 18.6 Mbp and 151 genes. The strongest candidate gene was FLNC because filamin C, the encoded protein, is muscle-specific and associated with myofibrillar myopathy. Sequencing of FLNC cDNA identified a c.752T>C (p.Met251Thr) mutation in the N-terminal actin-binding domain (ABD); this mutation segregated with the disease and was absent in 200 controls. We identified an Italian family with the same phenotype and found a c.577G>A (p.Ala193Thr) filamin C ABD mutation that segregated with the disease. Filamin C ABD mutations have not been described, although filamin A and filamin B ABD mutations cause multiple musculoskeletal disorders. The distal myopathy phenotype and muscle pathology in the two families differ from myofibrillar myopathies caused by filamin C rod and dimerization domain mutations because of the distinct involvement of hand muscles and lack of pathological protein aggregation. Thus, like the position of FLNA and B mutations, the position of the FLNC mutation determines disease phenotype. The two filamin C ABD mutations increase actin-binding affinity in a manner similar to filamin A and filamin B ABD mutations. Cell-culture expression of the c.752T>C (p.Met251)Thr mutant filamin C ABD demonstrated reduced nuclear localization as did mutant filamin A and filamin B ABDs. Expression of both filamin C ABD mutants as full-length proteins induced increased aggregation of filamin. We conclude filamin C ABD mutations cause a recognizable distal myopathy, most likely through increased actin affinity, similar to the pathological mechanism of filamin A and filamin B ABD mutations. PMID:21620354

  18. The carboxyterminal EF domain of erythroid α-spectrin is necessary for optimal spectrin-actin binding

    PubMed Central

    Korsgren, Catherine

    2010-01-01

    Spectrin and protein 4.1R crosslink F-actin, forming the membrane skeleton. Actin and 4.1R bind to one end of β-spectrin. The adjacent end of α-spectrin, called the EF domain, is calmodulin-like, with calcium-dependent and calcium-independent EF hands. The severely anemic sph1J/sph1J mouse has very fragile red cells and lacks the last 13 amino acids in the EF domain, implying that the domain is critical for skeletal integrity. To test this, we constructed a minispectrin heterodimer from the actin-binding domain, the EF domain, and 4 adjacent spectrin repeats in each chain. The minispectrin bound to F-actin in the presence of native human protein 4.1R. Formation of the spectrin-actin-4.1R complex was markedly attenuated when the minispectrin contained the shortened sph1J α-spectrin. The α-spectrin deletion did not interfere with spectrin heterodimer assembly or 4.1R binding but abolished the binary interaction between spectrin and F-actin. The data show that the α-spectrin EF domain greatly amplifies the function of the β-spectrin actin-binding domain (ABD) in forming the spectrin-actin-4.1R complex. A model, based on the structure of α-actinin, suggests that the EF domain modulates the function of the ABD and that the C-terminal EF hands (EF34) may bind to the linker that connects the ABD to the first spectrin repeat. PMID:20585040

  19. F-actin binding regions on the androgen receptor and huntingtin increase aggregation and alter aggregate characteristics.

    PubMed

    Angeli, Suzanne; Shao, Jieya; Diamond, Marc I

    2010-01-01

    Protein aggregation is associated with neurodegeneration. Polyglutamine expansion diseases such as spinobulbar muscular atrophy and Huntington disease feature proteins that are destabilized by an expanded polyglutamine tract in their N-termini. It has previously been reported that intracellular aggregation of these target proteins, the androgen receptor (AR) and huntingtin (Htt), is modulated by actin-regulatory pathways. Sequences that flank the polyglutamine tract of AR and Htt might influence protein aggregation and toxicity through protein-protein interactions, but this has not been studied in detail. Here we have evaluated an N-terminal 127 amino acid fragment of AR and Htt exon 1. The first 50 amino acids of ARN127 and the first 14 amino acids of Htt exon 1 mediate binding to filamentous actin in vitro. Deletion of these actin-binding regions renders the polyglutamine-expanded forms of ARN127 and Htt exon 1 less aggregation-prone, and increases the SDS-solubility of aggregates that do form. These regions thus appear to alter the aggregation frequency and type of polyglutamine-induced aggregation. These findings highlight the importance of flanking sequences in determining the propensity of unstable proteins to misfold. PMID:20140226

  20. Expression of Cofilin-1 and Transgelin in Esophageal Squamous Cell Carcinoma

    PubMed Central

    Zhang, Yan; Liao, Ruyi; Li, Hui; Liu, Ling; Chen, Xiao; Chen, Hongming

    2015-01-01

    Background Esophageal squamous cell carcinoma (ESCC) has attracted much research attention around the world, and the number of ESCC cases has increased gradually in recent years. Identifying the specific biomarkers of ESCC is an effective approach for the early diagnosis of tumors. Material/Methods Immunohistochemical streptavidin-peroxidase method was used to determine the expressions of Cofilin-1 and transgelin in 68 patients with esophageal squamous cell carcinoma (ESCC) and 48 individuals with normal esophageal tissues. In addition to the relationships between the expression of Cofilin-1 and transgelin, the clinicopathologic features of ESCC were also discussed. The correlation between Cofilin-1 and transgelin protein expression in ESCC was analyzed. Results (1) The positive expression rates of Cofilin-1 and transgelin were 60.3% (41/68) and 54.4% (37/68) in esophageal carcinoma tissue, respectively. The positive expression rates of Cofilin-1 and transgelin in normal esophageal tissue were 27.1% (13/48) and 29.1% (14/48), respectively. The differences were statistically significant (P<0.05). (2) The positive expression rate of Cofilin-1 did not differ significantly (P>0.05) with sex, age, ethnicity, tumor size, or infiltration depth; but did have a statistically significant (P<0.05) difference with various degrees of tumor differentiation, lymph node metastasis, and clinical stages. (3) The positive expression rate of transgelin did not differ significantly (P>0.05) with sex, age, ethnicity, tumor size, infiltration depth, and clinical stage, but did significantly (P<0.05) differ with degree of tumor differentiation and lymph node metastasis. Conclusions Cofilin-1 and transgelin may play roles in the carcinogenesis and development of esophageal squamous cell carcinoma. Cofilin-1 may be useful as an important biomarker for indicating the degree of malignancy of esophageal squamous cell carcinoma, and the detection of transgelin is valuable in early diagnosis of

  1. F-actin dismantling through a redox-driven synergy between Mical and cofilin.

    PubMed

    Grintsevich, Elena E; Yesilyurt, Hunkar Gizem; Rich, Shannon K; Hung, Ruei-Jiun; Terman, Jonathan R; Reisler, Emil

    2016-08-01

    Numerous cellular functions depend on actin filament (F-actin) disassembly. The best-characterized disassembly proteins, the ADF (actin-depolymerizing factor)/cofilins (encoded by the twinstar gene in Drosophila), sever filaments and recycle monomers to promote actin assembly. Cofilin is also a relatively weak actin disassembler, posing questions about mechanisms of cellular F-actin destabilization. Here we uncover a key link to targeted F-actin disassembly by finding that F-actin is efficiently dismantled through a post-translational-mediated synergism between cofilin and the actin-oxidizing enzyme Mical. We find that Mical-mediated oxidation of actin improves cofilin binding to filaments, where their combined effect dramatically accelerates F-actin disassembly compared with either effector alone. This synergism is also necessary and sufficient for F-actin disassembly in vivo, magnifying the effects of both Mical and cofilin on cellular remodelling, axon guidance and Semaphorin-Plexin repulsion. Mical and cofilin, therefore, form a redox-dependent synergistic pair that promotes F-actin instability by rapidly dismantling F-actin and generating post-translationally modified actin that has altered assembly properties. PMID:27454820

  2. Cofilin-2 Phosphorylation and Sequestration In Myocardial Aggregates: Novel Pathogenetic Mechanisms For Idiopathic Dilated Cardiomyopathy

    PubMed Central

    Subramanian, Khaushik; Gianni, Davide; Balla, Cristina; Assenza, Gabriele Egidy; Joshi, Mugdha; Semigran, Marc J.; Macgillivray, Thomas E.; Van Eyk, Jennifer E.; Agnetti, Giulio; Paolocci, Nazareno; Bamburg, James R.; Agrawal, Pankaj B.; del Monte, Federica

    2015-01-01

    BACKGROUND Recently, tangles and plaque-like aggregates have been identified in certain cases of dilated cardiomyopathy (DCM). This suggests a potential underlying cause for the one-third of cases, traditionally labeled idiopathic (iDCM), where there is no specific diagnostic test or targeted therapy. OBJECTIVE We sought to identify the make-up of myocardial aggregates to understand the molecular mechanisms of these cases of DCM; this strategy has been central to understanding Alzheimer’s disease. METHODS Aggregates were extracted from human iDCM samples with high congophilic reactivity (an indication of plaque presence) and the findings validated in a larger cohort of samples. We tested the expression, distribution, and activity of cofilin in human tissue and generated a cardiac-specific knockout mouse model to investigate the functional impact of the human findings. We also modeled cofilin inactivity in vitro using pharmacological and genetic gain and loss of function approaches. RESULTS Aggregates in the human myocardium were enriched for cofilin-2, an actin-depolymerizing protein known to participate in neurodegenerative diseases and nemaline myopathy. Cofilin-2 was predominantly phosphorylated, rendering it inactive. Cardiac-specific haploinsufficiency of cofilin-2 in mice recapitulated the human disease’s morphological, functional, and structural phenotype. Pharmacological stimulation of cofilin-2 phosphorylation and genetic overexpression of the phosphomimetic protein promoted the accumulation of “stress-like” fibers and severely impaired cardiomyocyte contractility. CONCLUSIONS Our study provides the first biochemical characterization of prefibrillar myocardial aggregates in humans and the first report to link cofilin-2 to cardiomyopathy. The findings suggest a common pathogenetic mechanism between certain iDCMs and other chronic degenerative diseases, laying the groundwork for new therapeutic strategies. PMID:25814227

  3. Tumor Suppressor Activity of Profilin Requires a Functional Actin Binding Site

    PubMed Central

    Wittenmayer, Nina; Jandrig, Burkhard; Rothkegel, Martin; Schlüter, Kathrin; Arnold, Wolfgang; Haensch, Wolfgang; Scherneck, Siegfried; Jockusch, Brigitte M.

    2004-01-01

    Profilin 1 (PFN1) is a regulator of the microfilament system and is involved in various signaling pathways. It interacts with many cytoplasmic and nuclear ligands. The importance of PFN1 for human tissue differentiation has been demonstrated by the findings that human cancer cells, expressing conspicuously low PFN1 levels, adopt a nontumorigenic phenotype upon raising their PFN1 level. In the present study, we characterize the ligand binding site crucial for profilin's tumor suppressor activity. Starting with CAL51, a human breast cancer cell line highly tumorigenic in nude mice, we established stable clones that express PFN1 mutants differentially defective in ligand binding. Clones expressing PFN1 mutants with reduced binding to either poly-proline-stretch ligands or phosphatidyl-inositol-4,5-bisphosphate, but with a functional actin binding site, were normal in growth, adhesion, and anchorage dependence, with only a weak tendency to elicit tumors in nude mice, similar to controls expressing wild-type PFN1. In contrast, clones expressing a mutant with severely reduced capacity to bind actin still behaved like the parental CAL51 and were highly tumorigenic. We conclude that the actin binding site on profilin is instrumental for normal differentiation of human epithelia and the tumor suppressor function of PFN1. PMID:14767055

  4. The N-terminal tropomyosin- and actin-binding sites are important for leiomodin 2's function.

    PubMed

    Ly, Thu; Moroz, Natalia; Pappas, Christopher T; Novak, Stefanie M; Tolkatchev, Dmitri; Wooldridge, Dayton; Mayfield, Rachel M; Helms, Gregory; Gregorio, Carol C; Kostyukova, Alla S

    2016-08-15

    Leiomodin is a potent actin nucleator related to tropomodulin, a capping protein localized at the pointed end of the thin filaments. Mutations in leiomodin-3 are associated with lethal nemaline myopathy in humans, and leiomodin-2-knockout mice present with dilated cardiomyopathy. The arrangement of the N-terminal actin- and tropomyosin-binding sites in leiomodin is contradictory and functionally not well understood. Using one-dimensional nuclear magnetic resonance and the pointed-end actin polymerization assay, we find that leiomodin-2, a major cardiac isoform, has an N-terminal actin-binding site located within residues 43-90. Moreover, for the first time, we obtain evidence that there are additional interactions with actin within residues 124-201. Here we establish that leiomodin interacts with only one tropomyosin molecule, and this is the only site of interaction between leiomodin and tropomyosin. Introduction of mutations in both actin- and tropomyosin-binding sites of leiomodin affected its localization at the pointed ends of the thin filaments in cardiomyocytes. On the basis of our new findings, we propose a model in which leiomodin regulates actin poly-merization dynamics in myocytes by acting as a leaky cap at thin filament pointed ends. PMID:27307584

  5. In vitro anti-cancer effects of the actin-binding natural compound rhizopodin.

    PubMed

    Zhang, S; Menche, D; Zahler, S; Vollmar, A M; Liebl, J; Förster, F

    2015-09-01

    Several natural compound interfere with microtubules or the actin cytoskeleton. Compounds interfering with the microtubules like Vinca-alkaloids or taxanes, are extensively used for cancer therapy. In contrast, knowledge about pharmacological properties of actin binding drugs is poor and drugs interfering with actin are far from clinical use. Rhizopodin is a natural compound that strongly affects the actin cytoskeleton at nanomolar concentrations. Initial work revealed interesting anti-bacterial and cytotoxic effects, but the cellular effects and pharmacological properties of rhizopodin have not been characterized. We hypothesized that rhizopodin might exert anti-cancer activity. Therefore, the aim of this study was to characterize the cellular and pharmacological effects of rhizopodin in cancer. Effects of rhizopodin demonstrated prominent effects on the actin cytoskeleton as shown in the actin-pyrene assay and by immunostaining of cancer cells. To investigate cellular effects of rhizopodin, we analyzed cell proliferation, cell death induction by propidium iodide exclusion and western blot, as well as migration by impedance measurement using the xCELLligence device in MDA-MB-231 breast cancer and T24 bladder cancer cell lines. Rhizopodin inhibited proliferation and induced cell death of MDA-MB-231 and T24 cells at nanomolar concentrations. PARP cleavage by rhizopodin suggests caspase-dependent cell death induction. Importantly, rhizopodin potently inhibited MDA-MB-231 and T24 cancer cell migration at subtoxic doses where no actin aggregation was observed, indicating a specific underlying signaling of rhizopodin. In summary, our study elucidates rhizopodin as actin-binding natural compound that exerts potent anti-cancer effects. Therefore, our work provides the basis for further in depth characterization of rhizopodin as an antitumoral agent. PMID:26492647

  6. Mutations in the cofilin partner Aip1/Wdr1 cause autoinflammatory disease and macrothrombocytopenia

    PubMed Central

    Panopoulos, Athanasia D.; Stirzaker, Roslynn A.; Hacking, Douglas F.; Tahtamouni, Lubna H.; Willson, Tracy A.; Mielke, Lisa A.; Henley, Katya J.; Zhang, Jian-Guo; Wicks, Ian P.; Stevenson, William S.; Nurden, Paquita; Watowich, Stephanie S.; Justice, Monica J.

    2007-01-01

    A pivotal mediator of actin dynamics is the protein cofilin, which promotes filament severing and depolymerization, facilitating the breakdown of existing filaments, and the enhancement of filament growth from newly created barbed ends. It does so in concert with actin interacting protein 1 (Aip1), which serves to accelerate cofilin's activity. While progress has been made in understanding its biochemical functions, the physiologic processes the cofilin/Aip1 complex regulates, particularly in higher organisms, are yet to be determined. We have generated an allelic series for WD40 repeat protein 1 (Wdr1), the mammalian homolog of Aip1, and report that reductions in Wdr1 function produce a dramatic phenotype gradient. While severe loss of function at the Wdr1 locus causes embryonic lethality, macrothrombocytopenia and autoinflammatory disease develop in mice carrying hypomorphic alleles. Macrothrombocytopenia is the result of megakaryocyte maturation defects, which lead to a failure of normal platelet shedding. Autoinflammatory disease, which is bone marrow–derived yet nonlymphoid in origin, is characterized by a massive infiltration of neutrophils into inflammatory lesions. Cytoskeletal responses are impaired in Wdr1 mutant neutrophils. These studies establish an essential requirement for Wdr1 in megakaryocytes and neutrophils, indicating that cofilin-mediated actin dynamics are critically important to the development and function of both cell types. PMID:17515402

  7. Drebrin-like protein DBN-1 is a sarcomere component that stabilizes actin filaments during muscle contraction.

    PubMed

    Butkevich, Eugenia; Bodensiek, Kai; Fakhri, Nikta; von Roden, Kerstin; Schaap, Iwan A T; Majoul, Irina; Schmidt, Christoph F; Klopfenstein, Dieter R

    2015-01-01

    Actin filament organization and stability in the sarcomeres of muscle cells are critical for force generation. Here we identify and functionally characterize a Caenorhabditis elegans drebrin-like protein DBN-1 as a novel constituent of the muscle contraction machinery. In vitro, DBN-1 exhibits actin filament binding and bundling activity. In vivo, DBN-1 is expressed in body wall muscles of C. elegans. During the muscle contraction cycle, DBN-1 alternates location between myosin- and actin-rich regions of the sarcomere. In contracted muscle, DBN-1 is accumulated at I-bands where it likely regulates proper spacing of α-actinin and tropomyosin and protects actin filaments from the interaction with ADF/cofilin. DBN-1 loss of function results in the partial depolymerization of F-actin during muscle contraction. Taken together, our data show that DBN-1 organizes the muscle contractile apparatus maintaining the spatial relationship between actin-binding proteins such as α-actinin, tropomyosin and ADF/cofilin and possibly strengthening actin filaments by bundling. PMID:26146072

  8. αT-Catenin Is a Constitutive Actin-binding α-Catenin That Directly Couples the Cadherin·Catenin Complex to Actin Filaments*

    PubMed Central

    Wickline, Emily D.; Dale, Ian W.; Merkel, Chelsea D.; Heier, Jonathon A.; Stolz, Donna B.

    2016-01-01

    α-Catenin is the primary link between the cadherin·catenin complex and the actin cytoskeleton. Mammalian αE-catenin is allosterically regulated: the monomer binds the β-catenin·cadherin complex, whereas the homodimer does not bind β-catenin but interacts with F-actin. As part of the cadherin·catenin complex, αE-catenin requires force to bind F-actin strongly. It is not known whether these properties are conserved across the mammalian α-catenin family. Here we show that αT (testes)-catenin, a protein unique to amniotes that is expressed predominantly in the heart, is a constitutive actin-binding α-catenin. We demonstrate that αT-catenin is primarily a monomer in solution and that αT-catenin monomer binds F-actin in cosedimentation assays as strongly as αE-catenin homodimer. The β-catenin·αT-catenin heterocomplex also binds F-actin with high affinity unlike the β-catenin·αE-catenin complex, indicating that αT-catenin can directly link the cadherin·catenin complex to the actin cytoskeleton. Finally, we show that a mutation in αT-catenin linked to arrhythmogenic right ventricular cardiomyopathy, V94D, promotes homodimerization, blocks β-catenin binding, and in cardiomyocytes disrupts localization at cell-cell contacts. Together, our data demonstrate that αT-catenin is a constitutively active actin-binding protein that can physically couple the cadherin·catenin complex to F-actin in the absence of tension. We speculate that these properties are optimized to meet the demands of cardiomyocyte adhesion. PMID:27231342

  9. Verprolin function in endocytosis and actin organization. Roles of the Las17p (yeast WASP)-binding domain and a novel C-terminal actin-binding domain.

    PubMed

    Thanabalu, Thirumaran; Rajmohan, Rajamuthiah; Meng, Lei; Ren, Gang; Vajjhala, Parimala R; Munn, Alan L

    2007-08-01

    Vrp1p (verprolin, End5p) is the yeast ortholog of human Wiskott-Aldrich syndrome protein (WASP)-interacting protein (WIP). Vrp1p localizes to the cortical actin cytoskeleton, is necessary for its polarization to sites of growth and is also essential for endocytosis. At elevated temperature, Vrp1p becomes essential for growth. A C-terminal Vrp1p fragment (C-Vrp1p) retains the ability to localize to the cortical actin cytoskeleton and function in actin-cytoskeleton polarization, endocytosis and growth. Here, we demonstrate that two submodules in C-Vrp1p are required for actin-cytoskeleton polarization: a novel C-terminal actin-binding submodule (CABS) that contains a novel G-actin-binding domain, which we call a verprolin homology 2 C-terminal (VH2-C) domain; and a second submodule comprising the Las17p-binding domain (LBD) that binds Las17p (yeast WASP). The LBD localizes C-Vrp1p to membranes and the cortical actin cytoskeleton. Intriguingly, the LBD is sufficient to restore endocytosis and growth at elevated temperature to Vrp1p-deficient cells. The CABS also restores these functions, but only if modified by a lipid anchor to provide membrane association. Our findings highlight the role of Las17p binding for Vrp1p membrane association, suggest general membrane association may be more important than specific targeting to the cortical actin cytoskeleton for Vrp1p function in endocytosis and cell growth, and suggest that Vrp1p binding to individual effectors may alter their physiological activity. PMID:17635585

  10. Importance of internal regions and the overall length of tropomyosin for actin binding and regulatory function.

    PubMed

    Hitchcock-DeGregori, S E; Song, Y; Moraczewska, J

    2001-02-20

    Tropomyosin (Tm) binds along actin filaments, one molecule spanning four to seven actin monomers, depending on the isoform. Periodic repeats in the sequence have been proposed to correspond to actin binding sites. To learn the functional importance of length and the internal periods we made a series of progressively shorter Tms, deleting from two up to six of the internal periods from rat striated alpha-TM (dAc2--3, dAc2--4, dAc3--5, dAc2--5, dAc2--6, dAc1.5--6.5). Recombinant Tms (unacetylated) were expressed in Escherichia coli. Tropomyosins that are four or more periods long (dAc2--3, dAc2--4, and dAc3--5) bound well to F-actin with troponin (Tn). dAc2--5 bound weakly (with EGTA) and binding of shorter mutants was undetectable in any condition. Myosin S1-induced binding of Tm to actin in the tight Tm-binding "open" state did not correlate with actin binding. dAc3--5 and dAc2--5 did not bind to actin even when the filament was saturated with S1. In contrast, dAc2--3 and dAc2--4 did, like wild-type-Tm, requiring about 3 mol of S1/mol of Tm for half-maximal binding. The results show the critical importance of period 5 (residues 166--207) for myosin S1-induced binding. The Tms that bound to actin (dAc2--3, dAc2--4, and dAc3--5) all fully inhibited the actomyosin ATPase (+Tn) in EGTA. In the presence of Ca(2+), relief of inhibition by these Tms was incomplete. We conclude (1) four or more actin periods are required for Tm to bind to actin with reasonable affinity and (2) that the structural requirements of Tm for the transition of the regulated filament from the blocked-to-closed/open (relief of inhibition by Ca(2+)) and the closed-to-open states (strong Tm binding to actin-S1) are different. PMID:11329279

  11. EWS represses cofilin 1 expression by inducing nuclear retention of cofilin 1 mRNA.

    PubMed

    Huang, L; Kuwahara, I; Matsumoto, K

    2014-06-01

    In Ewing's sarcoma family tumors (ESFTs), the proto-oncogene EWS that encodes an RNA-binding protein is fused by chromosomal translocation to the gene encoding one of the E-twenty six (ETS) family of transcription factors, most commonly friend leukemia virus integration 1 (FLI-1). Although EWS/FLI-1 chimeric proteins are necessary for carcinogenesis, additional events seem to be required for transformation to occur. We have previously reported that a protein product of an EWS mRNA target, whose expression is negatively regulated by EWS but not by EWS/FLI-1, contributes to ESFT development. However, the mechanism by which EWS represses protein expression remains to be elucidated. Here, we report that overexpression of full-length EWS repressed protein expression and induced nuclear retention of reporter mRNAs in a tethering assay. In contrast, when a mutant lacking the EWS C-terminal nuclear localization signal (classified as a PY-NLS) was expressed, reporter protein expression was upregulated, and the number of cells exporting reporter mRNA to the cytoplasm increased. EWS binds to the 3'-untranslated region in another mRNA target, cofilin 1 (CFL1), and negatively regulates the expression of CFL1. Overexpression of EWS induced nuclear retention of CFL1 mRNA. Furthermore, ESFT cell proliferation and metastatic potential were suppressed by small interfering RNA-mediated CFL1 knockdown. Together, our findings suggest that EWS induces nuclear retention of CFL1 mRNA, thereby suppressing expression of CFL1, and that CFL1 promotes development of ESFT. Targeting CFL1 might therefore provide another novel approach for treatment of this aggressive disease. PMID:23831569

  12. Cucurbitacin E as a new inhibitor of cofilin phosphorylation in human leukemia U937 cells.

    PubMed

    Nakashima, Souichi; Matsuda, Hisashi; Kurume, Ai; Oda, Yoshimi; Nakamura, Seikou; Yamashita, Masayuki; Yoshikawa, Masayuki

    2010-05-01

    Cucurbitane-type triterpenes, cucurbitacins B and E, were reported to exhibit cytotoxic effects in several cell lines mediated by JAK/STAT3 signaling. However, neither compound inhibited phosphorylation of STAT3 in human leukemia (U937) cells at low concentrations. We therefore synthesized a biotin-linked cucurbitacin E to isolate target proteins based on affinity for the molecule. As a result, cofilin, which regulates the depolymerization of actin, was isolated and suggested to be a target. Cucurbitacins E and I inhibited the phosphorylation of cofilin in a concentration-dependent manner, and their effective concentrations having the same range as the concentrations at which they had cytotoxic effects in U937 cells. In addition, the fibrous-/globular-actin ratio was decreased after treatment with cucurbitacin E in HT1080 cells. These findings suggested that the inhibition of cofilin's phosphorylation increased the severing activity of cofilin, and then the depolymerization of actin was enhanced after treatment with cucurbitacin E at lower concentrations. PMID:20347305

  13. Cofilin Phosphorylation Decreased by Serum-free Starvation with Low Glucose in the L6 Myoblasts.

    PubMed

    Kim, Mee-Young; Kim, Ju-Hyun; Lee, Jeong-Uk; Lee, Lim-Kyu; Yang, Seung-Min; Park, Byoung-Sun; Jeon, Hye-Joo; Lee, Won-Deok; Noh, Ji-Woong; Kwak, Taek-Yong; Jang, Sung-Ho; Lee, Tae-Hyun; Kim, Ju-Young; Kim, Tae-Whan; Kim, Bokyung; Kim, Junghwan

    2014-10-01

    [Purpose] Many studies have been using cell culture models of muscle cells with exogenous cytokines or glucocorticoids to mimic atrophy in in vivo and in vitro tests. However, the changes in the phosphorylation of atrophy-related cofilin are still poorly understood in starved skeletal muscle cells. In this study, we first examined whether or not phosphorylation of cofilin is altered in L6 myoblasts after 3, 6, 12, 24, 48, and 72 hours of serum-free starvation with low glucose. [Methods] We used Western blotting to exam protein expression and phosphorylation in atrophied L6 myoblasts. [Results] L6 cell sizes and numbers were diminished as a result of serum-free starvation in a time-dependent manner. Serum-free starvation for 3, 6, 12, 24, 48, and 72 hours significantly decreased the phosphorylation of cofilin, respectively. [Conclusion] These results suggest that starvation-induced atrophy may be in part related to changes in the phosphorylation of cofilin in L6 myoblasts. PMID:25364107

  14. Cyclic stretch promotes osteogenesis-related gene expression in osteoblast-like cells through a cofilin-associated mechanism.

    PubMed

    Gao, Jie; Fu, Shanmin; Zeng, Zhaobin; Li, Feifei; Niu, Qiannan; Jing, Da; Feng, Xue

    2016-07-01

    Osteoblasts have the capacity to perceive and transduce mechanical signals, and thus, regulate the mRNA and protein expression of a variety of genes associated with osteogenesis. Cytoskeletal reconstruction, as one of the earliest perception events for external mechanical stimulation, has previously been demonstrated to be essential for mechanotransduction in bone cells. However, the mechanism by which mechanical signals induce cytoskeletal deformation remains poorly understood. The actin‑binding protein, cofilin, promotes the depolymerization of actin and is understood to be important in the regulation of activities in various cell types, including endothelial, neuronal and muscle cells. However, to the best of our knowledge, the importance of cofilin in osteoblastic mechanotransduction has not been previously investigated. In the present study, osteoblast‑like MG‑63 cells were subjected to physiological cyclic stretch stimulation (12% elongation) for 1, 4, 8, 12 and 24 h, and the expression levels of cofilin and osteogenesis-associated genes were quantified with reverse transcription‑quantitative polymerase chain reaction, immunofluorescence staining and western blotting analyses. Additionally, knockdown of cofilin using RNA interference was conducted, and the mRNA levels of osteogenesis‑associated genes were compared between osteoblast‑like cells in the presence and absence of cofilin gene knockdown. The results of the present study demonstrated that cyclic stretch stimulates the expression of genes associated with osteoblastic activities in MG‑63 cells, including alkaline phosphatase (ALP), osteocalcin (OCN), runt‑related transcription factor 2 (Runx2) and collagen‑1 (COL‑1). Cyclic stretch also regulates the mRNA and protein expression of cofilin in MG‑63 cells. Furthermore, stretch‑induced increases in the levels of osteogenesis-associated genes, including ALP, OCN, Runx2 and COL‑1, were reduced following cofilin gene knockdown

  15. Methylmercury disrupts the balance between phosphorylated and non-phosphorylated cofilin in primary cultures of mice cerebellar granule cells A proteomic study

    SciTech Connect

    Vendrell, Iolanda; Carrascal, Montserrat; Abian, Joaquin

    2010-01-01

    Methylmercury is an environmental contaminant that is particularly toxic to the developing central nervous system; cerebellar granule neurons are especially vulnerable. Here, primary cultures of cerebellar granule cells (CGCs) were continuously exposed to methylmercury for up to 16 days in vitro (div). LC50 values were 508 +- 199, 345 +- 47, and 243 +- 45 nM after exposure for 6, 11, and 16 div, respectively. Proteins from cultured mouse CGCs were separated by 2DE. Seventy-one protein spots were identified by MALDI-TOF PMF and MALDI-TOF/TOF sequencing. Prolonged exposure to a subcytotoxic concentration of methylmercury significantly increased non-phosphorylated cofilin both in cell protein extracts (1.4-fold; p < 0.01) and in mitochondrial-enriched fractions (1.7-fold; p < 0.01). The decrease in P-cofilin induced by methylmercury was concentration-dependent and occurred after different exposure times. The percentage of P-cofilin relative to total cofilin significantly decreased to 49 +- 13% vs. control cells after exposure to 300 nM methylmercury for 5 div. The balance between the phosphorylated and non-phosphorylated form of cofilin regulates actin dynamics and facilitates actin filament turnover. Filamentous actin dynamics and reorganization are responsible of neuron shape change, migration, polarity formation, regulation of synaptic structures and function, and cell apoptosis. An alteration of the complex regulation of the cofilin phosphorylation/dephosphorylation pathway could be envisaged as an underlying mechanism compatible with reported signs of methylmercury-induced neurotoxicity.

  16. Formation of cofilin-actin rods following cucurbitacin-B-induced actin aggregation depends on Slingshot homolog 1-mediated cofilin hyperactivation.

    PubMed

    Zhang, Yan-Ting; Ouyang, Dong-Yun; Xu, Li-Hui; Zha, Qing-Bing; He, Xian-Hui

    2013-10-01

    Accumulating evidence indicates that cucurbitacin B (CuB), as well as other cucurbitacins, damages the actin cytoskeleton in a variety of cell types. However, the underlying mechanism of such an effect is not well understood. In this study, we showed that CuB rapidly induced actin aggregation followed by actin rod formation in melanoma cells. Cofilin, a critical regulator of actin dynamics, was dramatically dephosphorylated (i.e., activated) upon CuB treatment. Notably, the activated cofilin subsequently formed rod-like aggregates, which were highly colocalized with actin rods, indicating the formation of cofilin-actin rods. Cofilin knockdown significantly suppressed rod formation but did not prevent actin aggregation. Furthermore, knockdown of the cofilin phosphatase Slingshot homolog 1 (SSH1), but not chronophin (CIN), alleviated CuB-induced cofilin hyperactivation and cofilin-actin rod formation. The activity of Rho kinase and LIM kinase, two upstream regulators of cofilin activation, was downregulated after cofilin hyperactivation. Pretreatment with a thiol-containing reactive oxygen species (ROS) scavenger N-acetyl cysteine, but not other ROS inhibitors without thiol groups, suppressed CuB-induced actin aggregation, cofilin hyperactivation and cofilin-actin rod formation, suggesting that thiol oxidation might be involved in these processes. Taken together, our results demonstrated that CuB-induced formation of cofilin-actin rods was mediated by SSH1-dependent but CIN-independent cofilin hyperactivation. PMID:23695982

  17. A new role for cofilin in retinal neovascularization.

    PubMed

    Kumar, Raj; Janjanam, Jagadeesh; Singh, Nikhlesh K; Rao, Gadiparthi N

    2016-03-15

    Pak1 plays an important role in several cellular processes, including cell migration, but its role in pathological angiogenesis is not known. Here, we have determined its role in pathological retinal angiogenesis using an oxygen-induced retinopathy (OIR) model. VEGFA induced phosphorylation of Pak1 and its effector cofilin in a manner that was dependent on time as well as p38MAPKβ (also known as MAPK11) in human retinal microvascular endothelial cells (HRMVECs). Depletion of the levels of any of these molecules inhibited VEGFA-induced HRMVEC F-actin stress fiber formation, migration, proliferation, sprouting and tube formation. In accordance with these observations, hypoxia induced Pak1 and cofilin phosphorylation with p38MAPKβ being downstream to Pak1 and upstream to cofilin in mouse retina. Furthermore, Pak1 deficiency abolished hypoxia-induced p38MAPKβ and cofilin phosphorylation and abrogated retinal endothelial cell proliferation, tip cell formation and neovascularization. In addition, small interfering RNA (siRNA)-mediated downregulation of p38MAPKβ or cofilin levels in the wild-type mouse retina also diminished endothelial cell proliferation, tip cell formation and neovascularization. Taken together, these observations suggest that, although the p38MAPKβ-Pak1-cofilin axis is required for HRMVEC migration, proliferation, sprouting and tubulogenesis, Pak1-p38MAPKβ-cofilin signaling is also essential for hypoxia-induced mouse retinal endothelial cell proliferation, tip cell formation and neovascularization. PMID:26857814

  18. Identification of Arabidopsis Cyclase-associated Protein 1 as the First Nucleotide Exchange Factor for Plant Actin

    PubMed Central

    Chaudhry, Faisal; Guérin, Christophe; von Witsch, Matthias

    2007-01-01

    The actin cytoskeleton powers organelle movements, orchestrates responses to abiotic stresses, and generates an amazing array of cell shapes. Underpinning these diverse functions of the actin cytoskeleton are several dozen accessory proteins that coordinate actin filament dynamics and construct higher-order assemblies. Many actin-binding proteins from the plant kingdom have been characterized and their function is often surprisingly distinct from mammalian and fungal counterparts. The adenylyl cyclase-associated protein (CAP) has recently been shown to be an important regulator of actin dynamics in vivo and in vitro. The disruption of actin organization in cap mutant plants indicates defects in actin dynamics or the regulated assembly and disassembly of actin subunits into filaments. Current models for actin dynamics maintain that actin-depolymerizing factor (ADF)/cofilin removes ADP–actin subunits from filament ends and that profilin recharges these monomers with ATP by enhancing nucleotide exchange and delivery of subunits onto filament barbed ends. Plant profilins, however, lack the essential ability to stimulate nucleotide exchange on actin, suggesting that there might be a missing link yet to be discovered from plants. Here, we show that Arabidopsis thaliana CAP1 (AtCAP1) is an abundant cytoplasmic protein; it is present at a 1:3 M ratio with total actin in suspension cells. AtCAP1 has equivalent affinities for ADP– and ATP–monomeric actin (Kd ∼ 1.3 μM). Binding of AtCAP1 to ATP–actin monomers inhibits polymerization, consistent with AtCAP1 being an actin sequestering protein. However, we demonstrate that AtCAP1 is the first plant protein to increase the rate of nucleotide exchange on actin. Even in the presence of ADF/cofilin, AtCAP1 can recharge actin monomers and presumably provide a polymerizable pool of subunits to profilin for addition onto filament ends. In turnover assays, plant profilin, ADF, and CAP act cooperatively to promote flux of

  19. The Actin-binding Domain of Cortactin is Dynamic and Unstructured and Affects Lateral and Longitudinal Contacts in F-actin

    PubMed Central

    Shvetsov, Alexander; Berkane, Emir; Chereau, David; Dominguez, Roberto; Reisler, Emil

    2011-01-01

    Cortactin is an F-actin- and Arp2/3 complex-binding protein, implicated in the regulation of cytoskeleton dynamics and cortical actin-assembly. The actin-binding domain of cortactin consists of a 6.5 tandem repeat of a 37-amino acid sequence known as the cortactin repeat (residues 80-325). Using a combination of structure prediction, circular dichroism and cysteine crosslinking, we tested a recently published three-dimensional model of the cortactin molecule in which the cortactin repeat is folded as a globular helical domain (Zhang et al., 2007). We show that the cortactin repeat is unstructured in solution. Thus, wild type and mutant constructs of the cortactin repeat, containing pairs of cysteines at positions 112 and 246, 83 and 112, 83 and 246, and 83 and 306, could be readily crosslinked with reagents of varying lengths (0–9.6 Å). Using yeast actin cysteine mutants, we also show that cortactin inhibits disulfide and dibromobimane crosslinking across the lateral and longitudinal interfaces of actin subunits in the filament, suggesting a weakening of inter-subunits contacts. Our results are in disagreement with the proposed model of the cortactin molecule and have important implications for our understanding of cortactin regulation of cytoskeleton dynamics. PMID:19089942

  20. The actin-binding domain of cortactin is dynamic and unstructured and affects lateral and longitudinal contacts in F-actin.

    PubMed

    Shvetsov, Alexander; Berkane, Emir; Chereau, David; Dominguez, Roberto; Reisler, Emil

    2009-02-01

    Cortactin is an F-actin- and Arp2/3 complex-binding protein, implicated in the regulation of cytoskeleton dynamics and cortical actin-assembly. The actin-binding domain of cortactin consists of a 6.5 tandem repeat of a 37-amino acid sequence known as the cortactin repeat (residues 80-325). Using a combination of structure prediction, circular dichroism, and cysteine crosslinking, we tested a recently published three-dimensional model of the cortactin molecule in which the cortactin repeat is folded as a globular helical domain [Zhang et al., 2007, Mol Cell 27:197-213]. We show that the cortactin repeat is unstructured in solution. Thus, wild type and mutant constructs of the cortactin repeat, containing pairs of cysteines at positions 112 and 246, 83 and 112, 83 and 246, and 83 and 306, could be readily crosslinked with reagents of varying lengths (0-9.6 A). Using yeast actin cysteine mutants, we also show that cortactin inhibits disulfide and dibromobimane crosslinking across the lateral and longitudinal interfaces of actin subunits in the filament, suggesting a weakening of intersubunits contacts. Our results are in disagreement with the proposed model of the cortactin molecule and have important implications for our understanding of cortactin regulation of cytoskeleton dynamics. PMID:19089942

  1. WISp39 binds phosphorylated Coronin 1B to regulate Arp2/3 localization and Cofilin-dependent motility.

    PubMed

    Howell, Michael; Brickner, Howard; Delorme-Walker, Violaine D; Choi, Justin; Saffin, Jean-Michel; Miller, Daniel; Panopoulos, Andreas; DerMardirossian, Céline; Fotedar, Arun; Margolis, Robert L; Fotedar, Rati

    2015-03-30

    We previously identified Waf1 Cip1 stabilizing protein 39 (WISp39) as a binding partner for heat shock protein 90 (Hsp90). We now report that WISp39 has an essential function in the control of directed cell migration, which requires WISp39 interaction with Hsp90. WISp39 knockdown (KD) resulted in the loss of directional motility of mammalian cells and profound changes in cell morphology, including the loss of a single leading edge. WISp39 binds Coronin 1B, known to regulate the Arp2/3 complex and Cofilin at the leading edge. WISp39 preferentially interacts with phosphorylated Coronin 1B, allowing it to complex with Slingshot phosphatase (SSH) to dephosphorylate and activate Cofilin. WISp39 also regulates Arp2/3 complex localization at the leading edge. WISp39 KD-induced morphological changes could be rescued by overexpression of Coronin 1B together with a constitutively active Cofilin mutant. We conclude that WISp39 associates with Hsp90, Coronin 1B, and SSH to regulate Cofilin activation and Arp2/3 complex localization at the leading edge. PMID:25800056

  2. Ezrin self-association involves binding of an N-terminal domain to a normally masked C-terminal domain that includes the F-actin binding site.

    PubMed Central

    Gary, R; Bretscher, A

    1995-01-01

    Ezrin is a membrane-cytoskeletal linking protein that is concentrated in actin-rich surface structures. It is closely related to the microvillar proteins radixin and moesin and to the tumor suppressor merlin/schwannomin. Cell extracts contain ezrin dimers and ezrin-moesin heterodimers in addition to monomers. Truncated ezrin fusion proteins were assayed by blot overlay to determine which regions mediate self-association. Here we report that ezrin self-association occurs by head-to-tail joining of distinct N-terminal and C-terminal domains. It is likely that these domains, termed N- and C-ERMADs (ezrin-radixin-moesin association domain), are responsible for homotypic and heterotypic associations among ERM family members. The N-ERMAD of ezrin resided within amino acids 1-296; deletion of 10 additional residues resulted in loss of activity. The C-ERMAD was mapped to the last 107 amino acids of ezrin, residues 479-585. The two residues at the C-terminus were required for activity, and the region from 530-585 was insufficient. The C-ERMAD was masked in the native monomer. Exposure of this domain required unfolding ezrin with sodium dodecyl sulfate or expressing the domain as part of a truncated protein. Intermolecular association could not occur unless the C-ERMAD had been made accessible to its N-terminal partner. It can be inferred that dimerization in vivo requires an activation step that exposes this masked domain. The conformationally inaccessible C-terminal region included the F-actin binding site, suggesting that this activity is likewise regulated by masking. Images PMID:7579708

  3. Cofilin 1 activation prevents the defects in axon elongation and guidance induced by extracellular alpha-synuclein

    PubMed Central

    Tilve, Sharada; Difato, Francesco; Chieregatti, Evelina

    2015-01-01

    Impaired adult neurogenesis and axon traumatic injury participate in the severity of neurodegenerative diseases. Alpha-synuclein, a cytosolic protein involved in Parkinson’s disease, may be released from neurons, suggesting a role for excess secreted alpha-synuclein in the onset and spread of the pathology. Here we provide evidence that long term exposure of young neurons to extracellular alpha-synuclein hampers axon elongation and growth cone turning. We show that actin turnover and the rate of movement of actin waves along the axon are altered, due to alpha-synuclein-induced inactivation of cofilin. Upon laser disruption of microfilaments, healing of axons is favored by the increased phosphorylation of cofilin, however, at later time points; the defect in neurite extension prevails, being lost the regulation of cofilin activity. Importantly, overexpression of the active form of cofilin in neurons exposed to alpha-synuclein is able to restore the movement of actin waves, physiological axon elongation and growth cone turning. Our study reveals the molecular basis of alpha-synuclein-driven deficits in growth and migration of newborn neurons, and in elongation and regeneration of adult neurons. PMID:26558842

  4. RanBP9 at the intersection between cofilin and Aβ pathologies: rescue of neurodegenerative changes by RanBP9 reduction

    PubMed Central

    Woo, J A; Boggess, T; Uhlar, C; Wang, X; Khan, H; Cappos, G; Joly-Amado, A; De Narvaez, E; Majid, S; Minamide, L S; Bamburg, J R; Morgan, D; Weeber, E; Kang, D E

    2015-01-01

    Molecular pathways underlying the neurotoxicity and production of amyloid β protein (Aβ) represent potentially promising therapeutic targets for Alzheimer's disease (AD). We recently found that overexpression of the scaffolding protein RanBP9 increases Aβ production in cell lines and in transgenic mice while promoting cofilin activation and mitochondrial dysfunction. Translocation of cofilin to mitochondria and induction of cofilin–actin pathology require the activation/dephosphorylation of cofilin by Slingshot homolog 1 (SSH1) and cysteine oxidation of cofilin. In this study, we found that endogenous RanBP9 positively regulates SSH1 levels and mediates Aβ-induced translocation of cofilin to mitochondria and induction of cofilin–actin pathology in cultured cells, primary neurons, and in vivo. Endogenous level of RanBP9 was also required for Aβ-induced collapse of growth cones in immature neurons (days in vitro 9 (DIV9)) and depletion of synaptic proteins in mature neurons (DIV21). In vivo, amyloid precursor protein (APP)/presenilin-1 (PS1) mice exhibited 3.5-fold increased RanBP9 levels, and RanBP9 reduction protected against cofilin–actin pathology, synaptic damage, gliosis, and Aβ accumulation associated with APP/PS1 mice. Brains slices derived from APP/PS1 mice showed significantly impaired long-term potentiation (LTP), and RanBP9 reduction significantly enhanced paired pulse facilitation and LTP, as well as partially rescued contextual memory deficits associated with APP/PS1 mice. Therefore, these results underscore the critical importance of endogenous RanBP9 not only in Aβ accumulation but also in mediating the neurotoxic actions of Aβ at the level of synaptic plasticity, mitochondria, and cofilin–actin pathology via control of the SSH1-cofilin pathway in vivo. PMID:25741591

  5. New Host Factors Important for Respiratory Syncytial Virus (RSV) Replication Revealed by a Novel Microfluidics Screen for Interactors of Matrix (M) Protein*

    PubMed Central

    Kipper, Sarit; Hamad, Samar; Caly, Leon; Avrahami, Dorit; Bacharach, Eran; Jans, David A.; Gerber, Doron; Bajorek, Monika

    2015-01-01

    Although human respiratory syncytial virus (RSV) is the most common cause of bronchiolitis and pneumonia in infants and elderly worldwide, there is no licensed RSV vaccine or effective drug treatment available. The RSV Matrix protein plays key roles in virus life cycle, being found in the nucleus early in infection in a transcriptional inhibitory role, and later localizing in viral inclusion bodies before coordinating viral assembly and budding at the plasma membrane. In this study, we used a novel, high throughput microfluidics platform and custom human open reading frame library to identify novel host cell binding partners of RSV matrix. Novel interactors identified included proteins involved in host transcription regulation, the innate immunity response, cytoskeletal regulation, membrane remodeling, and cellular trafficking. A number of these interactions were confirmed by immunoprecipitation and cellular colocalization approaches. Importantly, the physiological significance of matrix interaction with the actin-binding protein cofilin 1, caveolae protein Caveolin 2, and the zinc finger protein ZNF502 was confirmed. siRNA knockdown of the host protein levels resulted in reduced RSV virus production in infected cells. These results have important implications for future antiviral strategies aimed at targets of RSV matrix in the host cell. PMID:25556234

  6. Specific Conserved C-terminal Amino Acids of Caenorhabditis elegans HMP-1/α-Catenin Modulate F-actin Binding Independently of Vinculin*

    PubMed Central

    Maiden, Stephanie L.; Harrison, Neale; Keegan, Jack; Cain, Brian; Lynch, Allison M.; Pettitt, Jonathan; Hardin, Jeff

    2013-01-01

    Stable intercellular adhesions formed through the cadherin-catenin complex are important determinants of proper tissue architecture and help maintain tissue integrity during morphogenetic movements in developing embryos. A key regulator of this stability is α-catenin, which connects the cadherin-catenin complex to the actin cytoskeleton. Although the C-terminal F-actin-binding domain of α-catenin has been shown to be crucial for its function, a more detailed in vivo analysis of discrete regions and residues required for actin binding has not been performed. Using Caenorhabditis elegans as a model system, we have characterized mutations in hmp-1/α-catenin that identify HMP-1 residues 687–742 and 826–927, as well as amino acid 802, as critical to the localization of junctional proximal actin during epidermal morphogenesis. We also find that the S823F transition in a hypomorphic allele, hmp-1(fe4), decreases actin binding in vitro. Using hmp-1(fe4) animals in a mutagenesis screen, we were then able to identify 11 intragenic suppressors of hmp-1(fe4) that revert actin binding to wild-type levels. Using homology modeling, we show that these amino acids are positioned at key conserved sites within predicted α-helices in the C terminus. Through the use of transgenic animals, we also demonstrate that HMP-1 residues 315–494, which correspond to a putative mechanotransduction domain that binds vinculin in vertebrate αE-catenin, are not required during epidermal morphogenesis but may aid efficient recruitment of HMP-1 to the junction. Our studies are the first to identify key conserved amino acids in the C terminus of α-catenin that modulate F-actin binding in living embryos of a simple metazoan. PMID:23271732

  7. Yeast mitochondria contain ATP-sensitive, reversible actin-binding activity.

    PubMed Central

    Lazzarino, D A; Boldogh, I; Smith, M G; Rosand, J; Pon, L A

    1994-01-01

    Sedimentation assays were used to demonstrate and characterize binding of isolated yeast mitochondria to phalloidin-stabilized yeast F-actin. These actin-mitochondrial interactions are ATP sensitive, saturable, reversible, and do not depend upon mitochondrial membrane potential. Protease digestion of mitochondrial outer membrane proteins or saturation of myosin-binding sites on F-actin with the S1 subfragment of skeletal myosin block binding. These observations indicate that a protein (or proteins) on the mitochondrial surface mediates ATP-sensitive, reversible binding of mitochondria to the lateral surface of microfilaments. Actin copurifies with mitochondria during subcellular fractionation and is released from the organelle upon treatment with ATP. Thus, actin-mitochondrial interactions resembling those observed in vitro may also exist in intact yeast cells. Finally, a yeast mutant bearing a temperature-sensitive mutation in the actin-encoding ACT1 gene (act1-3) displays temperature-dependent defects in transfer of mitochondria from mother cells to newly developed buds during yeast cell mitosis. Images PMID:7812049

  8. Inhibiting geranylgeranylation increases neurite branching and differentially activates cofilin in cell bodies and growth cones.

    PubMed

    Samuel, Filsy; Reddy, Jairus; Kaimal, Radhika; Segovia, Vianey; Mo, Huanbiao; Hynds, DiAnna L

    2014-08-01

    Inhibitors of the mevalonate pathway, including the highly prescribed statins, reduce the production of cholesterol and isoprenoids such as geranylgeranyl pyrophosphates. The Rho family of small guanine triphosphatases (GTPases) requires isoprenylation, specifically geranylgeranylation, for activation. Because Rho GTPases are primary regulators of actin filament rearrangements required for process extension, neurite arborization, and synaptic plasticity, statins may affect cognition or recovery from nervous system injury. Here, we assessed how manipulating geranylgeranylation affects neurite initiation, elongation, and branching in neuroblastoma growth cones. Treatment with the statin, lovastatin (20 μM), decreased measures of neurite initiation by 17.0 to 19.0 % when a source of cholesterol was present and increased neurite branching by 4.03- to 9.54-fold (regardless of exogenous cholesterol). Neurite elongation was increased by treatment with lovastatin only in cholesterol-free culture conditions. Treatment with lovastatin decreased growth cone actin filament content by up to 24.3 %. In all cases, co-treatment with the prenylation precursor, geranylgeraniol (10 μM), reversed the effect of lovastatin. In a prior work, statin effects on outgrowth were linked to modulating the actin depolymerizing factor, cofilin. In our assays, treatment with lovastatin or geranylgeraniol decreased cofilin phosphorylation in whole cell lysates. However, lovastatin increased cofilin phosphorylation in cell bodies and decreased it in growth cones, indicating differential regulation in specific cell regions. Together, we interpret these data to suggest that protein geranylgeranylation likely regulates growth cone actin filament content and subsequent neurite outgrowth through mechanisms that also affect actin nucleation and polymerization. PMID:24515839

  9. Cofilin/Twinstar Phosphorylation Levels Increase in Response to Impaired Coenzyme A Metabolism

    PubMed Central

    Siudeja, Katarzyna; Grzeschik, Nicola A.; Rana, Anil; de Jong, Jannie; Sibon, Ody C. M.

    2012-01-01

    Coenzyme A (CoA) is a pantothenic acid-derived metabolite essential for many fundamental cellular processes including energy, lipid and amino acid metabolism. Pantothenate kinase (PANK), which catalyses the first step in the conversion of pantothenic acid to CoA, has been associated with a rare neurodegenerative disorder PKAN. However, the consequences of impaired PANK activity are poorly understood. Here we use Drosophila and human neuronal cell cultures to show how PANK deficiency leads to abnormalities in F-actin organization. Cells with reduced PANK activity are characterized by abnormally high levels of phosphorylated cofilin, a conserved actin filament severing protein. The increased levels of phospho-cofilin coincide with morphological changes of PANK-deficient Drosophila S2 cells and human neuronal SHSY-5Y cells. The latter exhibit also markedly reduced ability to form neurites in culture – a process that is strongly dependent on actin remodeling. Our results reveal a novel and conserved link between a metabolic biosynthesis pathway, and regulation of cellular actin dynamics. PMID:22912811

  10. Neuroligin 1 regulates spines and synaptic plasticity via LIMK1/cofilin-mediated actin reorganization.

    PubMed

    Liu, An; Zhou, Zikai; Dang, Rui; Zhu, Yuehua; Qi, Junxia; He, Guiqin; Leung, Celeste; Pak, Daniel; Jia, Zhengping; Xie, Wei

    2016-02-15

    Neuroligin (NLG) 1 is important for synapse development and function, but the underlying mechanisms remain unclear. It is known that at least some aspects of NLG1 function are independent of the presynaptic neurexin, suggesting that the C-terminal domain (CTD) of NLG1 may be sufficient for synaptic regulation. In addition, NLG1 is subjected to activity-dependent proteolytic cleavage, generating a cytosolic CTD fragment, but the significance of this process remains unknown. In this study, we show that the CTD of NLG1 is sufficient to (a) enhance spine and synapse number, (b) modulate synaptic plasticity, and (c) exert these effects via its interaction with spine-associated Rap guanosine triphosphatase-activating protein and subsequent activation of LIM-domain protein kinase 1/cofilin-mediated actin reorganization. Our results provide a novel postsynaptic mechanism by which NLG1 regulates synapse development and function. PMID:26880202

  11. Epidermal Growth Factor Receptor-PI3K Signaling Controls Cofilin Activity To Facilitate Herpes Simplex Virus 1 Entry into Neuronal Cells

    PubMed Central

    Zheng, Kai; Xiang, Yangfei; Wang, Xiao; Wang, Qiaoli; Zhong, Meigong; Wang, Shaoxiang; Wang, Xiaoyan; Fan, Jianglin; Kitazato, Kaio; Wang, Yifei

    2014-01-01

    ABSTRACT Herpes simplex virus type 1 (HSV-1) establishes latency in neurons and can cause severe disseminated infection with neurological impairment and high mortality. This neurodegeneration is thought to be tightly associated with virus-induced cytoskeleton disruption. Currently, the regulation pattern of the actin cytoskeleton and the involved molecular mechanisms during HSV-1 entry into neurons remain unclear. Here, we demonstrate that the entry of HSV-1 into neuronal cells induces biphasic remodeling of the actin cytoskeleton and an initial inactivation followed by the subsequent activation of cofilin, a member of the actin depolymerizing factor family that is critical for actin reorganization. The disruption of F-actin dynamics or the modulation of cofilin activity by mutation, knockdown, or overexpression affects HSV-1 entry efficacy and virus-mediated cell ruffle formation. Binding of the HSV-1 envelope initiates the epidermal growth factor receptor (EGFR)-phosphatidylinositide 3-kinase (PI3K) signaling pathway, which leads to virus-induced early cofilin phosphorylation and F-actin polymerization. Moreover, the extracellular signal-regulated kinase (ERK) kinase and Rho-associated, coiled-coil-containing protein kinase 1 (ROCK) are recruited as downstream mediators of the HSV-1-induced cofilin inactivation pathway. Inhibitors specific for those kinases significantly reduce the virus infectivity without affecting virus binding to the target cells. Additionally, lipid rafts are clustered to promote EGFR-associated signaling cascade transduction. We propose that HSV-1 hijacks cofilin to initiate infection. These results could promote a better understanding of the pathogenesis of HSV-1-induced neurological diseases. PMID:24425731

  12. PTP1B-dependent regulation of receptor tyrosine kinase signaling by the actin-binding protein Mena.

    PubMed

    Hughes, Shannon K; Oudin, Madeleine J; Tadros, Jenny; Neil, Jason; Del Rosario, Amanda; Joughin, Brian A; Ritsma, Laila; Wyckoff, Jeff; Vasile, Eliza; Eddy, Robert; Philippar, Ulrike; Lussiez, Alisha; Condeelis, John S; van Rheenen, Jacco; White, Forest; Lauffenburger, Douglas A; Gertler, Frank B

    2015-11-01

    During breast cancer progression, alternative mRNA splicing produces functionally distinct isoforms of Mena, an actin regulator with roles in cell migration and metastasis. Aggressive tumor cell subpopulations express Mena(INV), which promotes tumor cell invasion by potentiating EGF responses. However, the mechanism by which this occurs is unknown. Here we report that Mena associates constitutively with the tyrosine phosphatase PTP1B and mediates a novel negative feedback mechanism that attenuates receptor tyrosine kinase signaling. On EGF stimulation, complexes containing Mena and PTP1B are recruited to the EGFR, causing receptor dephosphorylation and leading to decreased motility responses. Mena also interacts with the 5' inositol phosphatase SHIP2, which is important for the recruitment of the Mena-PTP1B complex to the EGFR. When Mena(INV) is expressed, PTP1B recruitment to the EGFR is impaired, providing a mechanism for growth factor sensitization to EGF, as well as HGF and IGF, and increased resistance to EGFR and Met inhibitors in signaling and motility assays. In sum, we demonstrate that Mena plays an important role in regulating growth factor-induced signaling. Disruption of this attenuation by Mena(INV) sensitizes tumor cells to low-growth factor concentrations, thereby increasing the migration and invasion responses that contribute to aggressive, malignant cell phenotypes. PMID:26337385

  13. Computational spatiotemporal analysis identifies WAVE2 and cofilin as joint regulators of costimulation-mediated T cell actin dynamics.

    PubMed

    Roybal, Kole T; Buck, Taráz E; Ruan, Xiongtao; Cho, Baek Hwan; Clark, Danielle J; Ambler, Rachel; Tunbridge, Helen M; Zhang, Jianwei; Verkade, Paul; Wülfing, Christoph; Murphy, Robert F

    2016-01-01

    Fluorescence microscopy is one of the most important tools in cell biology research because it provides spatial and temporal information to investigate regulatory systems inside cells. This technique can generate data in the form of signal intensities at thousands of positions resolved inside individual live cells. However, given extensive cell-to-cell variation, these data cannot be readily assembled into three- or four-dimensional maps of protein concentration that can be compared across different cells and conditions. We have developed a method to enable comparison of imaging data from many cells and applied it to investigate actin dynamics in T cell activation. Antigen recognition in T cells by the T cell receptor (TCR) is amplified by engagement of the costimulatory receptor CD28. We imaged actin and eight core actin regulators to generate over a thousand movies of T cells under conditions in which CD28 was either engaged or blocked in the context of a strong TCR signal. Our computational analysis showed that the primary effect of costimulation blockade was to decrease recruitment of the activator of actin nucleation WAVE2 (Wiskott-Aldrich syndrome protein family verprolin-homologous protein 2) and the actin-severing protein cofilin to F-actin. Reconstitution of WAVE2 and cofilin activity restored the defect in actin signaling dynamics caused by costimulation blockade. Thus, we have developed and validated an approach to quantify protein distributions in time and space for the analysis of complex regulatory systems. PMID:27095595

  14. NudC regulates actin dynamics and ciliogenesis by stabilizing cofilin 1.

    PubMed

    Zhang, Cheng; Zhang, Wen; Lu, Yi; Yan, Xiaoyi; Yan, Xiumin; Zhu, Xueliang; Liu, Wei; Yang, Yuehong; Zhou, Tianhua

    2016-02-01

    Emerging data indicate that actin dynamics is associated with ciliogenesis. However, the underlying mechanism remains unclear. Here we find that nuclear distribution gene C (NudC), an Hsp90 co-chaperone, is required for actin organization and dynamics. Depletion of NudC promotes cilia elongation and increases the percentage of ciliated cells. Further results show that NudC binds to and stabilizes cofilin 1, a key regulator of actin dynamics. Knockdown of cofilin 1 also facilitates ciliogenesis. Moreover, depletion of either NudC or cofilin 1 causes similar ciliary defects in zebrafish, including curved body, pericardial edema and defective left-right asymmetry. Ectopic expression of cofilin 1 significantly reverses the phenotypes induced by NudC depletion in both cultured cells and zebrafish. Thus, our data suggest that NudC regulates actin cytoskeleton and ciliogenesis by stabilizing cofilin 1. PMID:26704451

  15. Enhanced docetaxel-mediated cytotoxicity in human prostate cancer cells through knockdown of cofilin-1 by carbon nanohorn delivered siRNA.

    PubMed

    Pérez-Martínez, Francisco C; Carrión, Blanca; Lucío, Maria I; Rubio, Noelia; Herrero, María A; Vázquez, Ester; Ceña, Valentín

    2012-11-01

    We synthesized a non-viral delivery system (f-CNH3) for small interfering RNA (siRNA) by anchoring a fourth-generation polyamidoamine dendrimer (G4-PAMAM) to carbon nanohorns (CNHs). Using this new compound, we delivered a specific siRNA designed to knockdown cofilin-1, a key protein in the regulation of cellular cytoskeleton, to human prostate cancer (PCa) cells. The carbon nanohorn (CNH) derivative was able to bind siRNA and release it in the presence of an excess of the polyanion heparin. Moreover, this hybrid nanomaterial protected the siRNA from RNAse-mediated degradation. Synthetic siRNA delivered to PCa cells by f-CNH3 decreased the cofilin-1 mRNA and protein levels to about 20% of control values. Docetaxel, the drug of choice for the treatment of PCa, produced a concentration-dependent activation of caspase-3, an increase in cell death assessed by lactate dehydrogenase release to the culture medium, cell cycle arrest and inhibition of tumor cell proliferation. All of these toxic effects were potentiated when cofilin-1 was down regulated in these cells by a siRNA delivered by the nanoparticle. This suggests that knocking down certain proteins involved in cancer cell survival and/or proliferation may potentiate the cytotoxic actions of anticancer drugs and it might be a new therapeutic approach to treat tumors. PMID:22858003

  16. Cofilin Increases the Bending Flexibility of Actin Filaments: Implications for Severing and Cell Mechanics

    PubMed Central

    McCullough, Brannon R.; Blanchoin, Laurent; Martiel, Jean-Louis; De La Cruz, Enrique M.

    2009-01-01

    We determined the flexural (bending) rigidities of actin and cofilactin filaments from a cosine correlation function analysis of their thermally driven, two-dimensional fluctuations in shape. The persistence length of actin filaments is 9.8 µm, corresponding to a flexural rigidity of 0.040 pN µm2. Cofilin binding lowers the persistence length ∼5-fold to a value of 2.2 µm and the filament flexural rigidity to 0.0091 pN µm2. That cofilin-decorated filaments are more flexible than native filaments despite an increased mass indicates that cofilin binding weakens and redistributes stabilizing subunit interactions of filaments. We favor a mechanism in which the increased flexibility of cofilin-decorated filaments results from the linked dissociation of filament-stabilizing ions and reorganization of actin subdomain 2 and as a consequence promotes severing due to a mechanical asymmetry. Knowledge of the effects of cofilin on actin filament bending mechanics, together with our previous analysis of torsional stiffness, provide a quantitative measure of the mechanical changes in actin filaments associated with cofilin binding, and suggest that the overall mechanical and force-producing properties of cells can be modulated by cofilin activity. PMID:18617188

  17. The epidermal growth factor receptor regulates cofilin activity and promotes transmissible gastroenteritis virus entry into intestinal epithelial cells

    PubMed Central

    Hu, Weiwei; Zhu, Liqi; Yang, Xing; Lin, Jian; Yang, Qian

    2016-01-01

    Transmissible gastroenteritis virus (TGEV), a coronavirus, causes severe diarrhea and high mortality in newborn piglets. The porcine intestinal epithelium is the target of TGEV infection, but the mechanisms that TGEV disrupts the actin cytoskeleton and invades the host epithelium remain largely unknown. We not only found that TGEV infection stimulates F-actin to gather at the cell membrane but the disruption of F-actin inhibits TGEV entry as well. Cofilin is involved in F-actin reorganization and TGEV entry. The TGEV spike protein is capable of binding with EGFR, activating the downstream phosphoinositide-3 kinase (PI3K), then causing the phosphorylation of cofilin and F-actin polymerization via Rac1/Cdc42 GTPases. Inhibition of EGFR and PI3K decreases the entry of TGEV. EGFR is also the upstream activator of mitogen-activated protein kinase (MAPK) signaling pathways that is involved in F-actin reorganization. Additionally, lipid rafts act as signal platforms for the EGFR-associated signaling cascade and correlate with the adhesion of TGEV. In conlusion, these results provide valuable data of the mechanisms which are responsible for the TGEV pathogenesis and may lead to the development of new methods about controlling TGEV. PMID:26933809

  18. Proteomic Approaches Identify Members of Cofilin Pathway Involved in Oral Tumorigenesis

    PubMed Central

    Polachini, Giovana M.; Sobral, Lays M.; Mercante, Ana M. C.; Paes-Leme, Adriana F.; Xavier, Flávia C. A.; Henrique, Tiago; Guimarães, Douglas M.; Vidotto, Alessandra; Fukuyama, Erica E.; Góis-Filho, José F.; Cury, Patricia M.; Curioni, Otávio A.; Michaluart Jr, Pedro; Silva, Adriana M. A.; Wünsch-Filho, Victor; Nunes, Fabio D.; Leopoldino, Andréia M.; Tajara, Eloiza H.

    2012-01-01

    The prediction of tumor behavior for patients with oral carcinomas remains a challenge for clinicians. The presence of lymph node metastasis is the most important prognostic factor but it is limited in predicting local relapse or survival. This highlights the need for identifying biomarkers that may effectively contribute to prediction of recurrence and tumor spread. In this study, we used one- and two-dimensional gel electrophoresis, mass spectrometry and immunodetection methods to analyze protein expression in oral squamous cell carcinomas. Using a refinement for classifying oral carcinomas in regard to prognosis, we analyzed small but lymph node metastasis-positive versus large, lymph node metastasis-negative tumors in order to contribute to the molecular characterization of subgroups with risk of dissemination. Specific protein patterns favoring metastasis were observed in the “more-aggressive” group defined by the present study. This group displayed upregulation of proteins involved in migration, adhesion, angiogenesis, cell cycle regulation, anti-apoptosis and epithelial to mesenchymal transition, whereas the “less-aggressive” group was engaged in keratinocyte differentiation, epidermis development, inflammation and immune response. Besides the identification of several proteins not yet described as deregulated in oral carcinomas, the present study demonstrated for the first time the role of cofilin-1 in modulating cell invasion in oral carcinomas. PMID:23227181

  19. Jak3 Enables Chemokine-Dependent Actin Cytoskeleton Reorganization by Regulating Cofilin and Rac/Rhoa GTPases Activation

    PubMed Central

    Ambriz-Peña, Xochitl; García-Zepeda, Eduardo Alberto; Meza, Isaura; Soldevila, Gloria

    2014-01-01

    We have previously shown that Jak3 is involved in the signaling pathways of CCR7, CCR9 and CXCR4 in murine T lymphocytes and that Jak3−/− lymphocytes display an intrinsic defect in homing to peripheral lymph nodes. However, the molecular mechanism underlying the defective migration observed in Jak3−/− lymphocytes remains elusive. Here, it is demonstrated for the first time, that Jak3 is required for the actin cytoskeleton reorganization in T lymphocytes responding to chemokines. It was found that Jak3 regulates actin polymerization by controlling cofilin inactivation in response to CCL21 and CXCL12. Interestingly, cofilin inactivation was not precluded in PTX- treated cells despite their impaired actin polymerization. Additionally, Jak3 was required for small GTPases Rac1 and RhoA activation, which are indispensable for acquisition of the migratory cell phenotype and the generation of a functional leading edge and uropod, respectively. This defect correlates with data obtained by time-lapse video-microscopy showing an incompetent uropod formation and impaired motility in Jak3-pharmacologically inhibited T lymphocytes. Our data support a new model in which Jak3 and heterotrimeric G proteins can use independent, but complementary, signaling pathways to regulate actin cytoskeleton dynamics during cell migration in response to chemokines. PMID:24498424

  20. Ultrasensitivity in the Cofilin Signaling Module: A Mechanism for Tuning T Cell Responses

    PubMed Central

    Ramirez-Munoz, Rocio; Castro-Sánchez, Patricia; Roda-Navarro, Pedro

    2016-01-01

    Ultrasensitivity allows filtering weak activating signals and responding emphatically to small changes in stronger stimuli. In the presence of positive feedback loops, ultrasensitivity enables the existence of bistability, which convert graded stimuli into switch-like, sometimes irreversible, responses. In this perspective, we discuss mechanisms that can potentially generate a bistable response in the phosphorylation/dephosphorylation monocycle that regulates the activity of cofilin in dynamic actin networks. We pay particular attention to the phosphatase Slingshot-1 (SSH-1), which is involved in a reciprocal regulation and a positive feedback loop for cofilin activation. Based on these signaling properties and experimental evidences, we propose that bistability in the cofilin signaling module might be instrumental in T cell responses to antigenic stimulation. Initially, a switch-like response in the amount of active cofilin as a function of SSH-1 activation might assist in controlling the naïve T cell specificity and sensitivity. Second, high concentrations of active cofilin might endow antigen-experienced T cells with faster and more efficient responses. We discuss the cofilin function in the context of T cell receptor triggering and spatial regulation of plasma membrane signaling molecules. PMID:26925064

  1. Ultrasensitivity in the Cofilin Signaling Module: A Mechanism for Tuning T Cell Responses.

    PubMed

    Ramirez-Munoz, Rocio; Castro-Sánchez, Patricia; Roda-Navarro, Pedro

    2016-01-01

    Ultrasensitivity allows filtering weak activating signals and responding emphatically to small changes in stronger stimuli. In the presence of positive feedback loops, ultrasensitivity enables the existence of bistability, which convert graded stimuli into switch-like, sometimes irreversible, responses. In this perspective, we discuss mechanisms that can potentially generate a bistable response in the phosphorylation/dephosphorylation monocycle that regulates the activity of cofilin in dynamic actin networks. We pay particular attention to the phosphatase Slingshot-1 (SSH-1), which is involved in a reciprocal regulation and a positive feedback loop for cofilin activation. Based on these signaling properties and experimental evidences, we propose that bistability in the cofilin signaling module might be instrumental in T cell responses to antigenic stimulation. Initially, a switch-like response in the amount of active cofilin as a function of SSH-1 activation might assist in controlling the naïve T cell specificity and sensitivity. Second, high concentrations of active cofilin might endow antigen-experienced T cells with faster and more efficient responses. We discuss the cofilin function in the context of T cell receptor triggering and spatial regulation of plasma membrane signaling molecules. PMID:26925064

  2. Structure of the 34 kDa F-actin-bundling protein ABP34 from Dictyostelium discoideum.

    PubMed

    Kim, Min-Kyu; Kim, Ji-Hye; Kim, Ji-Sun; Kang, Sa-Ouk

    2015-09-01

    The crystal structure of the 34 kDa F-actin-bundling protein ABP34 from Dictyostelium discoideum was solved by Ca(2+)/S-SAD phasing and refined at 1.89 Å resolution. ABP34 is a calcium-regulated actin-binding protein that cross-links actin filaments into bundles. Its in vitro F-actin-binding and F-actin-bundling activities were confirmed by a co-sedimentation assay and transmission electron microscopy. The co-localization of ABP34 with actin in cells was also verified. ABP34 adopts a two-domain structure with an EF-hand-containing N-domain and an actin-binding C-domain, but has no reported overall structural homologues. The EF-hand is occupied by a calcium ion with a pentagonal bipyramidal coordination as in the canonical EF-hand. The C-domain structure resembles a three-helical bundle and superposes well onto the rod-shaped helical structures of some cytoskeletal proteins. Residues 216-244 in the C-domain form part of the strongest actin-binding sites (193-254) and exhibit a conserved sequence with the actin-binding region of α-actinin and ABP120. Furthermore, the second helical region of the C-domain is kinked by a proline break, offering a convex surface towards the solvent area which is implicated in actin binding. The F-actin-binding model suggests that ABP34 binds to the side of the actin filament and residues 216-244 fit into a pocket between actin subdomains -1 and -2 through hydrophobic interactions. These studies provide insights into the calcium coordination in the EF-hand and F-actin-binding site in the C-domain of ABP34, which are associated through interdomain interactions. PMID:26327373

  3. Cofilin1 Controls Transcolumnar Plasticity in Dendritic Spines in Adult Barrel Cortex

    PubMed Central

    Tsubota, Tadashi; Okubo-Suzuki, Reiko; Ohashi, Yohei; Tamura, Keita; Ogata, Koshin; Yaguchi, Masae; Matsuyama, Makoto; Inokuchi, Kaoru; Miyashita, Yasushi

    2015-01-01

    During sensory deprivation, the barrel cortex undergoes expansion of a functional column representing spared inputs (spared column), into the neighboring deprived columns (representing deprived inputs) which are in turn shrunk. As a result, the neurons in a deprived column simultaneously increase and decrease their responses to spared and deprived inputs, respectively. Previous studies revealed that dendritic spines are remodeled during this barrel map plasticity. Because cofilin1, a predominant regulator of actin filament turnover, governs both the expansion and shrinkage of the dendritic spine structure in vitro, it hypothetically regulates both responses in barrel map plasticity. However, this hypothesis remains untested. Using lentiviral vectors, we knocked down cofilin1 locally within layer 2/3 neurons in a deprived column. Cofilin1-knocked-down neurons were optogenetically labeled using channelrhodopsin-2, and electrophysiological recordings were targeted to these knocked-down neurons. We showed that cofilin1 knockdown impaired response increases to spared inputs but preserved response decreases to deprived inputs, indicating that cofilin1 dependency is dissociated in these two types of barrel map plasticity. To explore the structural basis of this dissociation, we then analyzed spine densities on deprived column dendritic branches, which were supposed to receive dense horizontal transcolumnar projections from the spared column. We found that spine number increased in a cofilin1-dependent manner selectively in the distal part of the supragranular layer, where most of the transcolumnar projections existed. Our findings suggest that cofilin1-mediated actin dynamics regulate functional map plasticity in an input-specific manner through the dendritic spine remodeling that occurs in the horizontal transcolumnar circuits. These new mechanistic insights into transcolumnar plasticity in adult rats may have a general significance for understanding reorganization of

  4. Simvastatin Attenuates Neuropathic Pain by Inhibiting the RhoA/LIMK/Cofilin Pathway.

    PubMed

    Qiu, Y; Chen, W Y; Wang, Z Y; Liu, F; Wei, M; Ma, C; Huang, Y G

    2016-09-01

    Neuropathic pain occurs due to deleterious changes in the nervous system caused by a lesion or dysfunction. Currently, neuropathic pain management is unsatisfactory and remains a challenge in clinical practice. Studies have suggested that actin cytoskeleton remodeling may be associated with neural plasticity and may involve a nociceptive mechanism. Here, we found that the RhoA/LIM kinase (LIMK)/cofilin pathway, which regulates actin dynamics, was activated after chronic constriction injury (CCI) of the sciatic nerve. Treatments that reduced RhoA/LIMK/cofilin pathway activity, including simvastatin, the Rho kinase inhibitor Y-27632, and the synthetic peptide Tat-S3, attenuated actin filament disruption in the dorsal root ganglion and CCI-induced neuropathic pain. Over-activation of the cytoskeleton caused by RhoA/LIMK/cofilin pathway activation may produce a scaffold for the trafficking of nociceptive signaling factors, leading to chronic neuropathic pain. Here, we found that simvastatin significantly decreased the ratio of membrane/cytosolic RhoA, which was significantly increased after CCI, by inhibiting the RhoA/LIMK/cofilin pathway. This effect was highly dependent on the function of the cytoskeleton as a scaffold for signal trafficking. We conclude that simvastatin attenuated neuropathic pain in rats subjected to CCI by inhibiting actin-mediated intracellular trafficking to suppress RhoA/LIMK/cofilin pathway activity. PMID:27216618

  5. Structure, Subunit Topology, and Actin-binding Activity of the Arp2/3 Complex from Acanthamoeba

    PubMed Central

    Mullins, R. Dyche; Stafford, Walter F.; Pollard, Thomas D.

    1997-01-01

    The Arp2/3 complex, first isolated from Acanthamoeba castellani by affinity chromatography on profilin, consists of seven polypeptides; two actinrelated proteins, Arp2 and Arp3; and five apparently novel proteins, p40, p35, p19, p18, and p14 (Machesky et al., 1994). The complex is homogeneous by hydrodynamic criteria with a Stokes' radius of 5.3 nm by gel filtration, sedimentation coefficient of 8.7 S, and molecular mass of 197 kD by analytical ultracentrifugation. The stoichiometry of the subunits is 1:1:1:1:1:1:1, indicating the purified complex contains one copy each of seven polypeptides. In electron micrographs, the complex has a bilobed or horseshoe shape with outer dimensions of ∼13 × 10 nm, and mathematical models of such a shape and size are consistent with the measured hydrodynamic properties. Chemical cross-linking with a battery of cross-linkers of different spacer arm lengths and chemical reactivities identify the following nearest neighbors within the complex: Arp2 and p40; Arp2 and p35; Arp3 and p35; Arp3 and either p18 or p19; and p19 and p14. By fluorescent antibody staining with anti-p40 and -p35, the complex is concentrated in the cortex of the ameba, especially in linear structures, possibly actin filament bundles, that lie perpendicular to the leading edge. Purified Arp2/3 complex binds actin filaments with a Kd of 2.3 μM and a stoichiometry of approximately one complex molecule per actin monomer. In electron micrographs of negatively stained samples, Arp2/3 complex decorates the sides of actin filaments. EDC/NHS cross-links actin to Arp3, p35, and a low molecular weight subunit, p19, p18, or p14. We propose structural and topological models for the Arp2/3 complex and suggest that affinity for actin filaments accounts for the localization of complex subunits to actinrich regions of Acanthamoeba. PMID:9015304

  6. Cofilin-induced unidirectional cooperative conformational changes in actin filaments revealed by high-speed atomic force microscopy

    PubMed Central

    Ngo, Kien Xuan; Kodera, Noriyuki; Katayama, Eisaku; Ando, Toshio; Uyeda, Taro QP

    2015-01-01

    High-speed atomic force microscopy was employed to observe structural changes in actin filaments induced by cofilin binding. Consistent with previous electron and fluorescence microscopic studies, cofilin formed clusters along actin filaments, where the filaments were 2-nm thicker and the helical pitch was ∼25% shorter, compared to control filaments. Interestingly, the shortened helical pitch was propagated to the neighboring bare zone on the pointed-end side of the cluster, while the pitch on the barbed-end side was similar to the control. Thus, cofilin clusters induce distinctively asymmetric conformational changes in filaments. Consistent with the idea that cofilin favors actin structures with a shorter helical pitch, cofilin clusters grew unidirectionally toward the pointed-end of the filament. Severing was often observed near the boundaries between bare zones and clusters, but not necessarily at the boundaries. DOI: http://dx.doi.org/10.7554/eLife.04806.001 PMID:25642645

  7. A small molecule inhibitor of tropomyosin dissociates actin binding from tropomyosin-directed regulation of actin dynamics

    PubMed Central

    Bonello, Teresa T.; Janco, Miro; Hook, Jeff; Byun, Alex; Appaduray, Mark; Dedova, Irina; Hitchcock-DeGregori, Sarah; Hardeman, Edna C.; Stehn, Justine R.; Böcking, Till; Gunning, Peter W.

    2016-01-01

    The tropomyosin family of proteins form end-to-end polymers along the actin filament. Tumour cells rely on specific tropomyosin-containing actin filament populations for growth and survival. To dissect out the role of tropomyosin in actin filament regulation we use the small molecule TR100 directed against the C terminus of the tropomyosin isoform Tpm3.1. TR100 nullifies the effect of Tpm3.1 on actin depolymerisation but surprisingly Tpm3.1 retains the capacity to bind F-actin in a cooperative manner. In vivo analysis also confirms that, in the presence of TR100, fluorescently tagged Tpm3.1 recovers normally into stress fibers. Assembling end-to-end along the actin filament is thereby not sufficient for tropomyosin to fulfil its function. Rather, regulation of F-actin stability by tropomyosin requires fidelity of information communicated at the barbed end of the actin filament. This distinction has significant implications for perturbing tropomyosin-dependent actin filament function in the context of anti-cancer drug development. PMID:26804624

  8. Phospholipase C and cofilin are required for carcinoma cell directionality in response to EGF stimulation

    PubMed Central

    Mouneimne, Ghassan; Soon, Lilian; DesMarais, Vera; Sidani, Mazen; Song, Xiaoyan; Yip, Shu-Chin; Ghosh, Mousumi; Eddy, Robert; Backer, Jonathan M.; Condeelis, John

    2004-01-01

    The epidermal growth factor (EGF)–induced increase in free barbed ends, resulting in actin polymerization at the leading edge of the lamellipodium in carcinoma cells, occurs as two transients: an early one at 1 min and a late one at 3 min. Our results reveal that phospholipase (PLC) is required for triggering the early barbed end transient. Phosphoinositide-3 kinase selectively regulates the late barbed end transient. Inhibition of PLC inhibits cofilin activity in cells during the early transient, delays the initiation of protrusions, and inhibits the ability of cells to sense a gradient of EGF. Suppression of cofilin, using either small interfering RNA silencing or function-blocking antibodies, selectively inhibits the early transient. Therefore, our results demonstrate that the early PLC and cofilin-dependent barbed end transient is required for the initiation of protrusions and is involved in setting the direction of cell movement in response to EGF. PMID:15337778

  9. Biophysical characterization of cofilin-induced extension-torsion coupling in actin filaments.

    PubMed

    Kim, Jae In; Kwon, Junpyo; Baek, Inchul; Na, Sungsoo

    2016-06-14

    Cofilin makes the actin filament flexible and thermally unstable by disassembling the filament and inducing bending and torsional compliance. Actin monomers bound to cofilin are able to chemically and mechanically interact in response to external forces. In this study, we performed two molecular dynamics tensile tests for actin and cofilactin filaments under identical conditions. Surprisingly, cofilactin filaments were found to be twisted, generating shear stress caused by torsion. Additionally, analysis by plane stress assumption indicated that the extension-torsion coupling effect increases the amount of principal stress by 10%. Using elasticity and solid mechanics theories, our study elucidates the role of cofilin in the disassembly of actin filaments under tensile forces. PMID:27143106

  10. MicroRNA-429 inhibits the migration and invasion of colon cancer cells by targeting PAK6/cofilin signaling.

    PubMed

    Tian, Xiangyang; Wei, Zibai; Wang, Jia; Liu, Ping; Qin, Yijun; Zhong, Meizuo

    2015-08-01

    MicroRNAs (miRs), a class of non-coding RNAs 18-25 nucleotides in length, can lead to mRNA degradation or inhibit protein translation by directly binding to the 3'-untranslational region (UTR) of their target mRNAs. The deregulation of miR-429 has been suggested to be involved in the development and progression of colon cancer. However, the detailed molecular mechanism involved remains to be determined. The aim of the present study was to investigate the role of miR-429 in the regulation of migration and invasion of colon cancer cells using RT-qPCR and western blotting. The results showed that the expression of miR-429 was reduced in colon cancer cell lines, when compared to a normal colon epithelial cell line. Treatment with DNA demethylation agent 5-aza-2'-deoxycytidine and histone deacetylase inhibitor phenylbutyrate (PBA), or transfection with the pre-miR-429 lentivirus plasmid led to the upregulation of miR-429 expression, as well as inhibition of migration and invasion in colon cancer cells. Investigation of the molecular mechanism showed that PAK6 was a novel target of miR-429, and the expression of PAK6 was upregulated in colon cancer tissues and cell lines, and was negatively regulated by miR-429 in colon cancer cells. Moreover, the cofilin signaling acted as a downstream effector of miR-429 in colon cancer cells. In conclusion, the results of the present study suggested that miR-429 inhibits the migration and invasion of colon cancer cells, partly at least, by mediating the expression of PAK6, as well as the activity of cofilin signaling. Therefore, miR-429 is as a potential molecular target for the treatment of colon cancer. PMID:26058485

  11. The 5-phosphatase OCRL mediates retrograde transport of the mannose 6-phosphate receptor by regulating a Rac1-cofilin signalling module

    PubMed Central

    van Rahden, Vanessa A.; Brand, Kristina; Najm, Juliane; Heeren, Joerg; Pfeffer, Suzanne R.; Braulke, Thomas; Kutsche, Kerstin

    2012-01-01

    Mutations in the OCRL gene encoding the phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) 5-phosphatase OCRL cause Lowe syndrome (LS), which is characterized by intellectual disability, cataracts and selective proximal tubulopathy. OCRL localizes membrane-bound compartments and is implicated in intracellular transport. Comprehensive analysis of clathrin-mediated endocytosis in fibroblasts of patients with LS did not reveal any difference in trafficking of epidermal growth factor, low density lipoprotein or transferrin, compared with normal fibroblasts. However, LS fibroblasts displayed reduced mannose 6-phosphate receptor (MPR)-mediated re-uptake of the lysosomal enzyme arylsulfatase B. In addition, endosome-to-trans Golgi network (TGN) transport of MPRs was decreased significantly, leading to higher levels of cell surface MPRs and their enrichment in enlarged, retromer-positive endosomes in OCRL-depleted HeLa cells. In line with the higher steady-state concentration of MPRs in the endosomal compartment in equilibrium with the cell surface, anterograde transport of the lysosomal enzyme, cathepsin D was impaired. Wild-type OCRL counteracted accumulation of MPR in endosomes in an activity-dependent manner, suggesting that PI(4,5)P2 modulates the activity state of proteins regulated by this phosphoinositide. Indeed, we detected an increased amount of the inactive, phosphorylated form of cofilin and lower levels of the active form of PAK3 upon OCRL depletion. Levels of active Rac1 and RhoA were reduced or enhanced, respectively. Overexpression of Rac1 rescued both enhanced levels of phosphorylated cofilin and MPR accumulation in enlarged endosomes. Our data suggest that PI(4,5)P2 dephosphorylation through OCRL regulates a Rac1-cofilin signalling cascade implicated in MPR trafficking from endosomes to the TGN. PMID:22907655

  12. Phylogenetic Patterns of Codon Evolution in the ACTIN-DEPOLYMERIZING FACTOR/COFILIN (ADF/CFL) Gene Family

    PubMed Central

    Roy-Zokan, Eileen M.; Dyer, Kelly A.; Meagher, Richard B.

    2015-01-01

    The actin-depolymerizing factor/cofilin (ADF/CFL) gene family encodes a diverse group of relatively small proteins. Once known strictly as modulators of actin filament dynamics, recent research has demonstrated that these proteins are involved in a variety of cellular processes, from signal transduction to the cytonuclear trafficking of actin. In both plant and animal lineages, expression patterns of paralogs in the ADF/CFL gene family vary among tissue types and developmental stages. In this study we use computational approaches to investigate the evolutionary forces responsible for the diversification of the ADF/CFL gene family. Estimating the rate of non-synonymous to synonymous mutations (dN/dS) across phylogenetic lineages revealed that the majority of ADF/CFL codon positions were under strong purifying selection, with rare episodic events of accelerated protein evolution. In both plants and animals these instances of accelerated evolution were ADF/CFL subclass specific, and all of the sites under selection were located in regions of the protein that could serve in new functional roles. We suggest these sites may have been important in the functional diversification of ADF/CFL proteins. PMID:26717562

  13. [Cytoskeletal actin and its associated proteins. Some examples in Protista].

    PubMed

    Guillén, N; Carlier, M F; Brugerolle, G; Tardieux, I; Ausseil, J

    1998-06-01

    Many processes, cell motility being an example, require cells to remodel the actin cytoskeleton in response to both intracellular and extracellular signals. Reorganization of the actin cytoskeleton involves the rapid disassembly and reassembly of actin filaments, a phenomenon regulated by the action of particular actin-binding proteins. In recent years, an interest in studying actin regulation in unicellular organisms has arisen. Parasitic protozoan are among these organisms and studies of the cytoskeleton functions of these protozoan are relevant related to either cell biology or pathogenicity. To discuss recent data in this field, a symposium concerning "Actin and actin-binding proteins in protists" was held on May 8-11 in Paris, France, during the XXXV meeting of the French Society of Protistology. As a brief summary of the symposium we report here findings concerning the in vitro actin dynamic assembly, as well as the characterization of several actin-binding proteins from the parasitic protozoan Entamoeba histolytica, Trichomonas vaginalis and Plasmodium knowlesi. In addition, localization of actin in non-pathogen protists such as Prorocentrum micans and Crypthecodinium cohnii is also presented. The data show that some actin-binding proteins facilitate organization of filaments into higher order structures as pseudopods, while others have regulatory functions, indicating very particular roles for actin-binding proteins. One of the proteins discussed during the symposium, the actin depolymerizing factor ADF, was shown to enhance the treadmilling rate of actin filaments. In vitro, ADF binds to the ADP-bound forms of G-actin and F-actin, thereby participating in and changing the rate of actin assembly. Biochemical approaches allowed the identification of a protein complex formed by HSP/C70-cap32-34 which might also be involved in depolymerization of F-actin in P. knowlesi. Molecular and cellular approaches were used to identify proteins such as ABP-120 and myosin

  14. The actin-binding protein profilin is required for germline stem cell maintenance and germ cell enclosure by somatic cyst cells

    PubMed Central

    Shields, Alicia R.; Spence, Allyson C.; Yamashita, Yukiko M.; Davies, Erin L.; Fuller, Margaret T.

    2014-01-01

    Specialized microenvironments, or niches, provide signaling cues that regulate stem cell behavior. In the Drosophila testis, the JAK-STAT signaling pathway regulates germline stem cell (GSC) attachment to the apical hub and somatic cyst stem cell (CySC) identity. Here, we demonstrate that chickadee, the Drosophila gene that encodes profilin, is required cell autonomously to maintain GSCs, possibly facilitating localization or maintenance of E-cadherin to the GSC-hub cell interface. Germline specific overexpression of Adenomatous Polyposis Coli 2 (APC2) rescued GSC loss in chic hypomorphs, suggesting an additive role of APC2 and F-actin in maintaining the adherens junctions that anchor GSCs to the niche. In addition, loss of chic function in the soma resulted in failure of somatic cyst cells to maintain germ cell enclosure and overproliferation of transit-amplifying spermatogonia. PMID:24346697

  15. Cyclic AMP-Rap1A signaling mediates cell surface translocation of microvascular smooth muscle α2C-adrenoceptors through the actin-binding protein filamin-2

    PubMed Central

    Motawea, Hanaa K. B.; Jeyaraj, Selvi C.; Eid, Ali H.; Mitra, Srabani; Unger, Nicholas T.; Ahmed, Amany A. E.; Flavahan, Nicholas A.

    2013-01-01

    The second messenger cyclic AMP (cAMP) plays a vital role in vascular physiology, including vasodilation of large blood vessels. We recently demonstrated cAMP activation of Epac-Rap1A and RhoA-Rho-associated kinase (ROCK)-F-actin signaling in arteriolar-derived smooth muscle cells increases expression and cell surface translocation of functional α2C-adrenoceptors (α2C-ARs) that mediate vasoconstriction in small blood vessels (arterioles). The Ras-related small GTPAse Rap1A increased expression of α2C-ARs and also increased translocation of perinuclear α2C-ARs to intracellular F-actin and to the plasma membrane. This study examined the mechanism of translocation to better understand the role of these newly discovered mediators of blood flow control, potentially activated in peripheral vascular disorders. We utilized a yeast two-hybrid screen with human microvascular smooth muscle cells (microVSM) cDNA library and the α2C-AR COOH terminus to identify a novel interaction with the actin cross-linker filamin-2. Yeast α-galactosidase assays, site-directed mutagenesis, and coimmunoprecipitation experiments in heterologous human embryonic kidney (HEK) 293 cells and in human microVSM demonstrated that α2C-ARs, but not α2A-AR subtype, interacted with filamin. In Rap1-stimulated human microVSM, α2C-ARs colocalized with filamin on intracellular filaments and at the plasma membrane. Small interfering RNA-mediated knockdown of filamin-2 inhibited Rap1-induced redistribution of α2C-ARs to the cell surface and inhibited receptor function. The studies suggest that cAMP-Rap1-Rho-ROCK signaling facilitates receptor translocation and function via phosphorylation of filamin-2 Ser2113. Together, these studies extend our previous findings to show that functional rescue of α2C-ARs is mediated through Rap1-filamin signaling. Perturbation of this signaling pathway may lead to alterations in α2C-AR trafficking and physiological function. PMID:23864608

  16. Transcriptional profiling of GBM invasion genes identifies effective inhibitors of the LIM kinase-Cofilin pathway

    PubMed Central

    Golbourn, Brian; Bertrand, Kelsey C.; Luck, Amanda; Sabha, Nesrin; Smith, Christian A.; Byron, Sara; Zadeh, Gelareh; Croul, Sidney; Berens, Michael; Rutka, James T.

    2014-01-01

    Malignant gliomas are highly proliferative and invasive neoplasms where total surgical resection is often impossible and effective local radiation therapy difficult. Consequently, there is a need to develop a greater understanding of the molecular events driving invasion and to identify novel treatment targets. Using microarray analysis comparing normal brain samples and mesenchymal glioblastoma multiforme (GBM), we identified over 140 significant genes involved in cell migration and invasion. The cofilin (CFL) pathway, which disassembles actin filaments, was highly up-regulated compared to normal brain. Up-regulation of LIM domain kinase 1 and 2 (LIMK1/2), that phosphorylates and inactivates cofilin, was confirmed in an additional independent data set comparing normal brain to GBM. We identified and utilized two small molecule inhibitors BMS-5 and Cucurbitacin I directed against the cofilin regulating kinases, LIMK1 and LIMK2, to target this pathway. Significant decreases in cell viability were observed in glioma cells treated with BMS-5 and Cucurbitacin I, while no cytotoxic effects were seen in normal astrocytes that lack LIMK. BMS-5 and Cucurbitacin I promoted increased adhesion in GBM cells, and decreased migration and invasion. Collectively, these data suggest that use of LIMK inhibitors may provide a novel way to target the invasive machinery in GBM. PMID:25237832

  17. Cofilin 1-Mediated Biphasic F-Actin Dynamics of Neuronal Cells Affect Herpes Simplex Virus 1 Infection and Replication

    PubMed Central

    Xiang, Yangfei; Zheng, Kai; Ju, Huaiqiang; Wang, Shaoxiang; Pei, Ying; Ding, Weichao; Chen, Zhenping; Wang, Qiaoli; Qiu, Xianxiu; Zhong, Meigong; Zeng, Fanli; Ren, Zhe; Qian, Chuiwen; Liu, Ge

    2012-01-01

    Herpes simplex virus 1 (HSV-1) invades the nervous system and causes pathological changes. In this study, we defined the remodeling of F-actin and its possible mechanisms during HSV-1 infection of neuronal cells. HSV-1 infection enhanced the formation of F-actin-based structures in the early stage of infection, which was followed by a continuous decrease in F-actin during the later stages of infection. The disruption of F-actin dynamics by chemical inhibitors significantly reduced the efficiency of viral infection and intracellular HSV-1 replication. The active form of the actin-depolymerizing factor cofilin 1 was found to increase at an early stage of infection and then to continuously decrease in a manner that corresponded to the remodeling pattern of F-actin, suggesting that cofilin 1 may be involved in the biphasic F-actin dynamics induced by HSV-1 infection. Knockdown of cofilin 1 impaired HSV-1-induced F-actin assembly during early infection and inhibited viral entry; however, overexpression of cofilin 1 did not affect F-actin assembly or viral entry during early infection but decreased intracellular viral reproduction efficiently. Our results, for the first time, demonstrated the biphasic F-actin dynamics in HSV-1 neuronal infection and confirmed the association of F-actin with the changes in the expression and activity of cofilin 1. These results may provide insight into the mechanism by which HSV-1 productively infects neuronal cells and causes pathogenesis. PMID:22623803

  18. Reactive oxygen species and hydrogen peroxide generation in cell migration

    PubMed Central

    Rudzka, Dominika A; Cameron, Jenifer M; Olson, Michael F

    2015-01-01

    Directional cell migration is a complex process that requires spatially and temporally co-ordinated regulation of actin cytoskeleton dynamics. In response to external cues, signals are transduced to elicit cytoskeletal responses. It has emerged that reactive oxygen species, including hydrogen peroxide, are important second messengers in pathways that influence the actin cytoskeleton, although the identities of key proteins regulated by hydrogen peroxide are largely unknown. We recently showed that oxidation of cofilin1 is elevated in migrating cells relative to stationary cells, and that the effect of this post-translational modification is to reduce cofilin1-actin binding and to inhibit filamentous-actin severing by cofilin1. These studies revealed that cofilin1 regulation by hydrogen peroxide contributes to directional cell migration, and established a template for discovering additional proteins that are regulated in an analogous manner. PMID:27066166

  19. Aggregation of Actin and Cofilin in Identical Twins with Juvenile-Onset Dystonia

    PubMed Central

    Gearing, Marla; Juncos, Jorge L.; Procaccio, Vincent; Gutekunst, Claire-Anne; Marino-Rodriguez, Elaine M.; Gyure, Kymberly A.; Ono, Shoichiro; Santoianni, Robert; Krawiecki, Nicolas S.; Wallace, Douglas C.; Wainer, Bruce H.

    2005-01-01

    The neuropathology of the primary dystonias is not well understood. We examined brains from identical twins with DYT1-negative, dopa-unresponsive dystonia. The twins exhibited mild developmental delays until age 12 years when they began developing rapidly progressive generalized dystonia. Genetic, metabolic, and imaging studies ruled out known causes of dystonia. Cognition was subnormal but stable until the last few years. Death occurred at ages 21 and 22 years. The brains were macroscopically unremarkable. Microscopic examination showed unusual glial fibrillary acidic protein–immunoreactive astrocytes in multiple regions and iron accumulation in pallidal and nigral neurons. However, the most striking findings were 1) eosinophilic, rod-like cytoplasmic inclusions in neocortical and thalamic neurons that were actin depolymerizing factor/cofilin-immunoreactive but only rarely actin-positive; and 2) abundant eosinophilic spherical structures in the striatum that were strongly actin- and actin depolymerizing factor/cofilin-positive. Electron microscopy suggested that these structures represent degenerating neurons and processes; the accumulating filaments had the same dimensions as actin microfilaments. To our knowledge, aggregation of actin has not been reported previously as the predominant feature in any neurodegenerative disease. Thus, our findings may shed light on a novel neuropathological change associated with dystonia that may represent a new degenerative mechanism involving actin, a ubiquitous constituent of the cytoskeletal system. PMID:12325076

  20. Structure and Function of a G-actin Sequestering Protein with a Vital Role in Malaria Oocyst Development inside the Mosquito Vector*

    PubMed Central

    Hliscs, Marion; Sattler, Julia M.; Tempel, Wolfram; Artz, Jennifer D.; Dong, Aiping; Hui, Raymond; Matuschewski, Kai; Schüler, Herwig

    2010-01-01

    Cyclase-associated proteins (CAPs) are evolutionary conserved G-actin-binding proteins that regulate microfilament turnover. CAPs have a modular structure consisting of an N-terminal adenylate cyclase binding domain, a central proline-rich segment, and a C-terminal actin binding domain. Protozoan parasites of the phylum Apicomplexa, such as Cryptosporidium and the malaria parasite Plasmodium, express small CAP orthologs with homology to the C-terminal actin binding domain (C-CAP). Here, we demonstrate by reverse genetics that C-CAP is dispensable for the pathogenic Plasmodium blood stages. However, c-cap(-) parasites display a complete defect in oocyst development in the insect vector. By trans-species complementation we show that the Cryptosporidium parvum ortholog complements the Plasmodium gene functions. Purified recombinant C. parvum C-CAP protein binds actin monomers and prevents actin polymerization. The crystal structure of C. parvum C-CAP shows two monomers with a right-handed β-helical fold intercalated at their C termini to form the putative physiological dimer. Our results reveal a specific vital role for an apicomplexan G-actin-binding protein during sporogony, the parasite replication phase that precedes formation of malaria transmission stages. This study also exemplifies how Plasmodium reverse genetics combined with biochemical and structural analyses of orthologous proteins can offer a fast track toward systematic gene characterization in apicomplexan parasites. PMID:20083609

  1. KCC2 Gates Activity-Driven AMPA Receptor Traffic through Cofilin Phosphorylation.

    PubMed

    Chevy, Quentin; Heubl, Martin; Goutierre, Marie; Backer, Stéphanie; Moutkine, Imane; Eugène, Emmanuel; Bloch-Gallego, Evelyne; Lévi, Sabine; Poncer, Jean Christophe

    2015-12-01

    Expression of the neuronal K/Cl transporter KCC2 is tightly regulated throughout development and by both normal and pathological neuronal activity. Changes in KCC2 expression have often been associated with altered chloride homeostasis and GABA signaling. However, recent evidence supports a role of KCC2 in the development and function of glutamatergic synapses through mechanisms that remain poorly understood. Here we show that suppressing KCC2 expression in rat hippocampal neurons precludes long-term potentiation of glutamatergic synapses specifically by preventing activity-driven membrane delivery of AMPA receptors. This effect is independent of KCC2 transporter function and can be accounted for by increased Rac1/PAK- and LIMK-dependent cofilin phosphorylation and actin polymerization in dendritic spines. Our results demonstrate that KCC2 plays a critical role in the regulation of spine actin cytoskeleton and gates long-term plasticity at excitatory synapses in cortical neurons. PMID:26631461

  2. The role of cofilin-l in vulvar squamous cell carcinoma: A marker of carcinogenesis, progression and targeted therapy.

    PubMed

    Wu, Qiong; Jiang, Ying; Cui, Shoubin; Wang, Yanshi; Wu, Xin

    2016-05-01

    Numerous studies have revealed that cofilin-l (CFL1) is associated with cancer cell migration and invasion in various types of tumor tissues. We investigated the roles of CFL1 in vulvar squamous cell carcinoma (VSCC). CFL1 expression was detected in VSCC and normal vulvar tissues using immunohistochemistry and western blotting. The vulvar carcinoma SW962 cell line was transfected with CFL1 small interfering RNA (siRNA) and exposed to periplocoside. We then assessed changes in cell proliferation, apoptosis, invasion and metastasis. We detected changes in CFL1 mRNA and protein expression by RT-PCR and western blotting, and alterations in protein expression of various relevant molecules by western blotting. CFL1 expression was found to be significantly upregulated in the VSCC tissues compared with the normal vulvar tissues by immunohistochemistry and western blotting (P<0.05) and was positively correlated with International Federation of Gynecology and Obstetrics (FIGO) stage, differentiation and lymphatic metastasis (P<0.05). After CFL1 knockdown by siRNA transfection, SW962 cells exhibited a decrease in growth, G1 phase cell cycle arrest, induction of apoptotic, low invasion and metastasis, and disrupted lamellipodium formation. We found that the protein expression of Bcl-xL, cyclin A1, MMP2, MMP9 and STAT3 was decreased, while expression of Bax was increased. Periplocoside inhibited SW962 cell growth, promoted apoptosis, suppressed invasion and migration, and lamellipodium formation. Periplocoside exposure resulted in lower CFL1, Bcl-xL, cyclin A1, MMP2, MMP9 and STAT3 levels, but a higher Bax level compared with the control group. We demonstrated that abnormal CFL1 expression may affect vulvar carcinogenesis and subsequent progression. CFL1 silencing by siRNA significantly inhibited VSCC cell progression, which suggests that CFL1 is a potential therapeutic target for vulvar cancer. Periplocoside, which was utilized in the present study for the clinical

  3. Dephosphorylation and mitochondrial translocation of cofilin sensitizes human leukemia cells to cerulenin-induced apoptosis via the ROCK1/Akt/JNK signaling pathway

    PubMed Central

    Liu, Yanxia; Li, Jing; Zhang, Hongwei; Hu, Xiaoye; Chen, Yibiao; Liu, Xin; Li, Yunong; Li, Ping; Liu, Ehu; Gao, Ning

    2016-01-01

    In this study, we determined that cerulenin, a natural product inhibitor of fatty acid synthase, induces mitochondrial injury and apoptosis in human leukemia cells through the mitochondrial translocation of cofilin. Only dephosphorylated cofilin could translocate to mitochondria during cerulenin-induced apoptosis. Disruption of the ROCK1/Akt/JNK signaling pathway plays a critical role in the cerulenin-mediated dephosphorylation and mitochondrial translocation of cofilin and apoptosis. In vivo studies demonstrated that cerulenin-mediated inhibition of tumor growth in a mouse xenograft model of leukemia was associated with mitochondrial translocation of cofilin and apoptosis. These data are consistent with a hierarchical model in which induction of apoptosis by cerulenin primarily results from activation of ROCK1, inactivation of Akt, and activation of JNK. This leads to the dephosphorylation and mitochondrial translocation of cofilin and culminates with cytochrome c release, caspase activation, and apoptosis. Our study has revealed a novel role of cofilin in the regulation of mitochondrial injury and apoptosis and suggests that cerulenin is a potential drug for the treatment of leukemia. PMID:26967395

  4. Actin Polymerization is Stimulated by Actin Crosslinking Protein Palladin

    PubMed Central

    Gurung, Ritu; Yadav, Rahul; Brungardt, Joseph G.; Orlova, Albina; Egelman, Edward H.; Beck, Moriah R.

    2016-01-01

    The actin scaffold protein palladin regulates both normal cell migration and invasive cell motility, processes that require the coordinated regulation of actin dynamics. However, the potential effect of palladin on actin dynamics has remained elusive. Here we show that the actin binding immunoglobulin-like domain of palladin, which is directly responsible for both actin binding and bundling, also stimulates actin polymerization in vitro. Palladin eliminated the lag phase that is characteristic of the slow nucleation step of actin polymerization. Furthermore, palladin dramatically reduced depolymerization, slightly enhanced the elongation rate, and did not alter the critical concentration. Microscopy and in vitro crosslinking assays reveal differences in actin bundle architecture when palladin is incubated with actin before or after polymerization. These results suggest a model whereby palladin stimulates a polymerization-competent form of G-actin, akin to metal ions, either through charge neutralization or conformational changes. PMID:26607837

  5. Two distinct domains of protein 4.1 critical for assembly of functional nuclei in vitro.

    PubMed

    Krauss, Sharon Wald; Heald, Rebecca; Lee, Gloria; Nunomura, Wataru; Gimm, J Aura; Mohandas, Narla; Chasis, Joel Anne

    2002-11-15

    Protein 4.1R, a multifunctional structural protein, acts as an adaptor in mature red cell membrane skeletons linking spectrin-actin complexes to plasma membrane-associated proteins. In nucleated cells protein 4.1 is not associated exclusively with plasma membrane but is also detected at several important subcellular locations crucial for cell division. To identify 4.1 domains having critical functions in nuclear assembly, 4.1 domain peptides were added to Xenopus egg extract nuclear reconstitution reactions. Morphologically disorganized, replication deficient nuclei assembled when spectrin-actin-binding domain or NuMA-binding C-terminal domain peptides were present. However, control variant spectrin-actin-binding domain peptides incapable of binding actin or mutant C-terminal domain peptides with reduced NuMA binding had no deleterious effects on nuclear reconstitution. To test whether 4.1 is required for proper nuclear assembly, 4.1 isoforms were depleted with spectrin-actin binding or C-terminal domain-specific antibodies. Nuclei assembled in the depleted extracts were deranged. However, nuclear assembly could be rescued by the addition of recombinant 4.1R. Our data establish that protein 4.1 is essential for nuclear assembly and identify two distinct 4.1 domains, initially characterized in cytoskeletal interactions, that have crucial and versatile functions in nuclear assembly. PMID:12171917

  6. Two distinct domains of protein 4.1 critical for assembly offunctional nuclei in Vitro

    SciTech Connect

    Krauss, Sharon Wald; Heald, Rebecca; Lee, Gloria; Nunomura, Wataru; Gimm,J. Aura; Mohandas, Narla; Chasis, Joel AnneJ. Aura; Mohandas, Narla; Chasis, Joel Anne

    2002-11-15

    Protein 4.1R, a multifunctional structural protein, acts asan adaptor in mature red cell membrane skeletons linking spectrin-actincomplexes to plasma membrane-associated proteins. In nucleated cellsprotein 4.1 is not associated exclusively with plasma membrane but isalso detected at several important subcellular locations crucial for celldivision. To identify 4.1 domains having critical functions in nuclearassembly, 4.1 domain peptides were added to Xenopus egg extract nuclearreconstitution reactions. Morphologically disorganized, replicationdeficient nuclei assembled when spectrin-actin binding domain orNuMA-binding C-terminal domain peptides were present. However, controlvariant spectrin-actin binding domain peptides incapable of bindingactin, or mutant C-terminal domain peptides with reduced NuMA binding,had no deleterious effects on nuclear reconstitution. To test if 4.1 isrequired for proper nuclear assembly, 4.1 isoforms were depleted withspectrin-actin binding or C-terminal domain-specific antibodies. Nucleiassembled in depleted extracts ha d deranged phenotypes. However, nuclearassembly could be rescued by addition of recombinant 4.1R. Our dataestablishes that protein 4.1 is essential for nuclear assembly andidentifies two distinct 4.1 domains, initially characterized incytoskeletal interactions, that have crucial and versatile functions innuclear assembly.

  7. VASP Activation via the Gα13/RhoA/PKA Pathway Mediates Cucurbitacin-B-Induced Actin Aggregation and Cofilin-Actin Rod Formation

    PubMed Central

    Zhang, Yan-Ting; Xu, Li-Hui; Lu, Qun; Liu, Kun-Peng; Liu, Pei-Yan; Ji, Fang; Liu, Xiao-Ming; Ouyang, Dong-Yun; He, Xian-Hui

    2014-01-01

    Cucurbitacin B (CuB), a potent antineoplastic agent of cucurbitacin triterpenoids, induces rapid disruption of actin cytoskeleton and aberrant cell cycle inhibiting carcinogenesis. However, the underlying molecular mechanism of such anticancer effects remains incompletely understood. In this study, we showed that CuB treatment rapidly induced vasodilator-stimulated phosphoprotein (VASP) phosphorylation (i.e. activation) at the Ser157 residue and generated VASP clumps which were co-localized with amorphous actin aggregates prior to the formation of highly-ordered cofilin-actin rods in melanoma cells. Knockdown of VASP or inhibition of VASP activation using PKA-specific inhibitor H89 suppressed CuB-induced VASP activation, actin aggregation and cofilin-actin rod formation. The VASP activation was mediated by cAMP-independent PKA activation as CuB decreased the levels of cAMP while MDL12330A, an inhibitor of adenylyl cyclase, had weak effect on VASP activation. Knockdown of either Gα13 or RhoA not only suppressed VASP activation, but also ameliorated CuB-induced actin aggregation and abrogated cofilin-actin rod formation. Collectively, our studies highlighted that the CuB-induced actin aggregation and cofilin-actin rod formation was mediated via the Gα13/RhoA/PKA/VASP pathway. PMID:24691407

  8. The Rho-GTPase effector ROCK regulates meiotic maturation of the bovine oocyte via myosin light chain phosphorylation and cofilin phosphorylation.

    PubMed

    Lee, So-Rim; Xu, Yong-Nan; Jo, Yu-Jin; Namgoong, Suk; Kim, Nam-Hyung

    2015-11-01

    Oocyte meiosis involves a unique asymmetric division involving spindle movement from the central cytoplasm to the cortex, followed by polar body extrusion. ROCK is a Rho-GTPase effector involved in various cellular functions in somatic cells as well as oocyte meiosis. ROCK was previously shown to promote actin organization by phosphorylating several downstream targets, including LIM domain kinase (LIMK), phosphorylated cofilin (p-cofilin), and myosin light chain (MLC). In this study, we investigated the roles of ROCK and MLC during bovine oocyte meiosis. We found that ROCK was localized around the nucleus at the oocyte's germinal-vesicle (GV) stage, but spreads to the rest of the cytoplasm in later developmental stages. On the other hand, phosphorylated MLC (p-MLC) localized at the cortex, and its abundance decreased by the metaphase-II stage. Disrupting ROCK activity, via RNAi or the chemical inhibitor Y-27632, blocked both cell cycle progression and polar body extrusion. ROCK inhibition also resulted in decreased cortical actin, p-cofilin, and p-MLC levels. Similar to the phenotype associated with inhibition of ROCK activity, inhibition of MLC kinase by the chemical inhibitor ML-7 caused defects in polar body extrusion. Collectively, our results suggest that the ROCK/MLC/actomyosin as well as ROCK/LIMK/cofilin pathways regulate meiotic spindle migration and cytokinesis during bovine oocyte maturation. PMID:26175189

  9. Actin polymerization is stimulated by actin cross-linking protein palladin.

    PubMed

    Gurung, Ritu; Yadav, Rahul; Brungardt, Joseph G; Orlova, Albina; Egelman, Edward H; Beck, Moriah R

    2016-02-15

    The actin scaffold protein palladin regulates both normal cell migration and invasive cell motility, processes that require the co-ordinated regulation of actin dynamics. However, the potential effect of palladin on actin dynamics has remained elusive. In the present study, we show that the actin-binding immunoglobulin-like domain of palladin, which is directly responsible for both actin binding and bundling, also stimulates actin polymerization in vitro. Palladin eliminated the lag phase that is characteristic of the slow nucleation step of actin polymerization. Furthermore, palladin dramatically reduced depolymerization, slightly enhanced the elongation rate, and did not alter the critical concentration. Microscopy and in vitro cross-linking assays reveal differences in actin bundle architecture when palladin is incubated with actin before or after polymerization. These results suggest a model whereby palladin stimulates a polymerization-competent form of globular or monomeric actin (G-actin), akin to metal ions, either through charge neutralization or through conformational changes. PMID:26607837

  10. A Mechanism for Actin Filament Severing by Malaria Parasite Actin Depolymerizing Factor 1 via a Low Affinity Binding Interface*

    PubMed Central

    Wong, Wilson; Webb, Andrew I.; Olshina, Maya A.; Infusini, Giuseppe; Tan, Yan Hong; Hanssen, Eric; Catimel, Bruno; Suarez, Cristian; Condron, Melanie; Angrisano, Fiona; NebI, Thomas; Kovar, David R.; Baum, Jake

    2014-01-01

    Actin depolymerizing factor (ADF)/cofilins are essential regulators of actin turnover in eukaryotic cells. These multifunctional proteins facilitate both stabilization and severing of filamentous (F)-actin in a concentration-dependent manner. At high concentrations ADF/cofilins bind stably to F-actin longitudinally between two adjacent actin protomers forming what is called a decorative interaction. Low densities of ADF/cofilins, in contrast, result in the optimal severing of the filament. To date, how these two contrasting modalities are achieved by the same protein remains uncertain. Here, we define the proximate amino acids between the actin filament and the malaria parasite ADF/cofilin, PfADF1 from Plasmodium falciparum. PfADF1 is unique among ADF/cofilins in being able to sever F-actin but do so without stable filament binding. Using chemical cross-linking and mass spectrometry (XL-MS) combined with structure reconstruction we describe a previously overlooked binding interface on the actin filament targeted by PfADF1. This site is distinct from the known binding site that defines decoration. Furthermore, total internal reflection fluorescence (TIRF) microscopy imaging of single actin filaments confirms that this novel low affinity site is required for F-actin severing. Exploring beyond malaria parasites, selective blocking of the decoration site with human cofilin (HsCOF1) using cytochalasin D increases its severing rate. HsCOF1 may therefore also use a decoration-independent site for filament severing. Thus our data suggest that a second, low affinity actin-binding site may be universally used by ADF/cofilins for actin filament severing. PMID:24371134

  11. Reorganization of actin filaments by ADF/cofilin is involved in formation of microtubule structures during Xenopus oocyte maturation

    PubMed Central

    Yamagishi, Yuka; Abe, Hiroshi

    2015-01-01

    We examined the reorganization of actin filaments and microtubules during Xenopus oocyte maturation. Surrounding the germinal vesicle (GV) in immature oocytes, the cytoplasmic actin filaments reorganized to accumulate beneath the vegetal side of the GV, where the microtubule-organizing center and transient microtubule array (MTOC-TMA) assembled, just before GV breakdown (GVBD). Immediately after GVBD, both Xenopus ADF/cofilin (XAC) and its phosphatase Slingshot (XSSH) accumulated into the nuclei and intranuclear actin filaments disassembled from the vegetal side with the shrinkage of the GV. As the MTOC-TMA developed well, cytoplasmic actin filaments were retained at the MTOC-TMA base region. Suppression of XAC dephosphorylation by anti-XSSH antibody injection inhibited both actin filament reorganization and proper formation and localization of both the MTOC-TMA and meiotic spindles. Stabilization of actin filaments by phalloidin also inhibited formation of the MTOC-TMA and disassembly of intranuclear actin filaments without affecting nuclear shrinkage. Nocodazole also caused the MTOC-TMA and the cytoplasmic actin filaments at its base region to disappear, which further impeded disassembly of intranuclear actin filaments from the vegetal side. XAC appears to reorganize cytoplasmic actin filaments required for precise assembly of the MTOC and, together with the MTOC-TMA, regulate the intranuclear actin filament disassembly essential for meiotic spindle formation. PMID:26424802

  12. Proteomic analysis of differentiating neuroblastoma cells treated with sub-lethal neurite inhibitory concentrations of diazinon: Identification of novel biomarkers of effect

    SciTech Connect

    Harris, W.; Sachana, M.; Flaskos, J.; Hargreaves, A.J.

    2009-10-15

    In previous work we showed that sub-lethal levels of diazinon inhibited neurite outgrowth in differentiating N2a neuroblastoma cells. Western blotting analysis targeted at proteins involved in axon growth and stress responses, revealed that such exposure led to a reduction in the levels of neurofilament heavy chain, microtubule associated protein 1 B (MAP 1B) and HSP-70. The aim of this study was to apply the approach of 2 dimensional polyacrylamide gel electrophoresis and mass spectrometry to identify novel biomarkers of effect. A number of proteins were found to be up-regulated compared to the control on silver-stained gels. These were classified in to 3 main groups of proteins: cytosolic factors, chaperones and the actin-binding protein cofilin, all of which are involved in cell differentiation, survival or metabolism. The changes observed for cofilin were further confirmed by quantitative Western blotting analysis with anti-actin and anti-cofilin antibodies. Indirect immunofluorescence staining with the same antibodies indicated that the microfilament network was disrupted in diazinon-treated cells. Our data suggest that microfilament organisation is disrupted by diazinon exposure, which may be related to increased cofilin expression.

  13. Cytoskeletal protein filamin A is a nucleolar protein that suppresses ribosomal RNA gene transcription.

    PubMed

    Deng, Wensheng; Lopez-Camacho, Cesar; Tang, Jen-Yang; Mendoza-Villanueva, Daniel; Maya-Mendoza, Apolinar; Jackson, Dean A; Shore, Paul

    2012-01-31

    Filamin A (FLNA) is an actin-binding protein with a well-established role in the cytoskeleton, where it determines cell shape and locomotion by cross-linking actin filaments. Mutations in FLNA are associated with a wide range of genetic disorders. Here we demonstrate a unique role for FLNA as a nucleolar protein that associates with the RNA polymerase I (Pol I) transcription machinery to suppress rRNA gene transcription. We show that depletion of FLNA by siRNAs increased rRNA expression, rDNA promoter activity and cell proliferation. Immunodepletion of FLNA from nuclear extracts resulted in a decrease in rDNA promoter-driven transcription in vitro. FLNA coimmunoprecipitated with the Pol I components actin, TIF-IA, and RPA40, and their occupancy of the rDNA promoter was increased in the absence of FLNA in vivo. The FLNA actin-binding domain is essential for the suppression of rRNA expression and for inhibiting recruitment of the Pol I machinery to the rDNA promoter. These findings reveal an additional role for FLNA as a regulator of rRNA gene expression and have important implications for our understanding of the role of FLNA in human disease. PMID:22307607

  14. Wdr1-mediated cell shape dynamics and cortical tension are essential for epidermal planar cell polarity

    PubMed Central

    Pasolli, H. Amalia; Chai, Sophia; Nikolova, Maria; Stokes, Nicole; Fuchs, Elaine

    2015-01-01

    During mouse development, core planar cell polarity (PCP) proteins become polarized in the epidermal plane to guide angling/morphogenesis of hair follicles. How PCP is established is poorly understood. Here, we identify a key role for Wdr1 (also known as Aip1), an F-actin-binding protein that enhances cofilin/destrin-mediated F-actin disassembly. We show that cofilin and destrin function redundantly in developing epidermis, but their combined depletion perturbs cell adhesion, cytokinesis, apicobasal polarity and PCP. Although Wdr1 depletion accentuates single-loss-of-cofilin/destrin phenotypes, alone it resembles core PCP mutations. Seeking a mechanism, we find that Wdr1 and cofilin/destrin-mediated actomyosin remodelling are essential for generating or maintaining cortical tension within the developing epidermal sheet and driving the cell shape and planar orientation changes that accompany establishment of PCP in mammalian epidermis. Our findings suggest intriguing evolutionary parallels but mechanistic modifications to the distal wing hinge-mediated mechanical forces that drive cell shape change and orient PCP in the Drosophila wing disc. PMID:25915128

  15. Skeletal Muscle MicroRNA and Messenger RNA Profiling in Cofilin-2 Deficient Mice Reveals Cell Cycle Dysregulation Hindering Muscle Regeneration

    PubMed Central

    Morton, Sarah U.; Joshi, Mugdha; Savic, Talia; Beggs, Alan H.; Agrawal, Pankaj B.

    2015-01-01

    Congenital myopathies are rare skeletal muscle diseases presenting in early age with hypotonia and weakness often linked to a genetic defect. Mutations in the gene for cofilin-2 (CFL2) have been identified in several families as a cause of congenital myopathy with nemaline bodies and cores. Here we explore the global messenger and microRNA expression patterns in quadriceps muscle samples from cofillin-2-null mice and compare them with sibling-matched wild-type mice to determine the molecular pathways and mechanisms involved. Cell cycle processes are markedly dysregulated, with altered expression of genes involved in mitotic spindle formation, and evidence of loss of cell cycle checkpoint regulation. Importantly, alterations in cell cycle, apoptosis and proliferation pathways are present in both mRNA and miRNA expression patterns. Specifically, p21 transcript levels were increased, and the expression of p21 targets, such as cyclin D and cyclin E, was decreased. We therefore hypothesize that deficiency of cofilin-2 is associated with interruption of the cell cycle at several checkpoints, hindering muscle regeneration. Identification of these pathways is an important step towards developing appropriate therapies against various congenital myopathies. PMID:25874796

  16. ADF and Cofilin1 Control Actin Stress Fibers, Nuclear Integrity, and Cell Survival

    PubMed Central

    Kanellos, Georgios; Zhou, Jing; Patel, Hitesh; Ridgway, Rachel A.; Huels, David; Gurniak, Christine B.; Sandilands, Emma; Carragher, Neil O.; Sansom, Owen J.; Witke, Walter; Brunton, Valerie G.; Frame, Margaret C.

    2015-01-01

    Summary Genetic co-depletion of the actin-severing proteins ADF and CFL1 triggers catastrophic loss of adult homeostasis in multiple tissues. There is impaired cell-cell adhesion in skin keratinocytes with dysregulation of E-cadherin, hyperproliferation of differentiated cells, and ultimately apoptosis. Mechanistically, the primary consequence of depleting both ADF and CFL1 is uncontrolled accumulation of contractile actin stress fibers associated with enlarged focal adhesions at the plasma membrane, as well as reduced rates of membrane protrusions. This generates increased intracellular acto-myosin tension that promotes nuclear deformation and physical disruption of the nuclear lamina via the LINC complex that normally connects regulated actin filaments to the nuclear envelope. We therefore describe a pathway involving the actin-severing proteins ADF and CFL1 in regulating the dynamic turnover of contractile actin stress fibers, and this is vital to prevent the nucleus from being damaged by actin contractility, in turn preserving cell survival and tissue homeostasis. PMID:26655907

  17. Evidence for an uncommon alpha-actinin protein in Trichomonas vaginalis.

    PubMed

    Bricheux, G; Coffe, G; Pradel, N; Brugerolle, G

    1998-09-15

    As part of our ongoing project of identification of actin-binding proteins implicated in the cell transition (flagellate to amoeboid/adherent) of Trichomonas vaginalis, we have characterized an alpha-actinin-related protein in this parasite. The protein (P100) has a molecular mass of 100 kDa and an isoelectric point of 5.5. A monoclonal antibody raised against this protein co-localizes with the actin network. P100 gene transcripts are co-expressed with actin throughout the cell cycle. Analysis of the deduced protein sequence reveals three domains: an N-terminal actin-binding region; a central region rich in alpha-helix; and a C-terminal domain with Ca(2+)-binding capacity. Whereas the N- and C-terminal regions are well-conserved as compared to other alpha-actinins, we observe in the central region an atypical distribution of residues in five repeats. The sequence of the repeats does not show any homology with the rod domain of the other alpha-actinins, except for the first repeat which shows some similarity. The four other repeats of T. vaginalis P100 appear to result from a duplication event which is not detectable in the other sequences. PMID:9803416

  18. Acanthamoeba castellanii: proteins involved in actin dynamics, glycolysis, and proteolysis are regulated during encystation.

    PubMed

    Bouyer, Sabrina; Rodier, Marie-Hélène; Guillot, Alain; Héchard, Yann

    2009-09-01

    Acanthamoeba castellanii is a pathogenic free-living amoeba. Cyst forms are particularly important in their pathogenicity, as they are more resistant to treatments and might protect pathogenic intracellular bacteria. However, encystation is poorly understood at the molecular level and global changes at the protein level have not been completely described. In this study, we performed two-dimensional gel electrophoresis to compare protein expression in trophozoite and cyst forms. Four proteins, specifically expressed in trophozoites, and four proteins, specifically expressed in cysts, were identified. Two proteins, enolase and fructose bisphosphate aldolase, are involved in the glycolytic pathway. Three proteins are likely actin-binding proteins, which is consistent with the dramatic morphological modifications of the cells during encystation. One protein belongs to the serine protease family and has been already linked to encystation in A. castellanii. In conclusion, this study found that the proteins whose expression was modified during encystation were likely involved in actin dynamics, glycolysis, and proteolysis. PMID:19523468

  19. Protein-protein interaction network analysis of cirrhosis liver disease

    PubMed Central

    Safaei, Akram; Rezaei Tavirani, Mostafa; Arefi Oskouei, Afsaneh; Zamanian Azodi, Mona; Mohebbi, Seyed Reza; Nikzamir, Abdol Rahim

    2016-01-01

    Aim: Evaluation of biological characteristics of 13 identified proteins of patients with cirrhotic liver disease is the main aim of this research. Background: In clinical usage, liver biopsy remains the gold standard for diagnosis of hepatic fibrosis. Evaluation and confirmation of liver fibrosis stages and severity of chronic diseases require a precise and noninvasive biomarkers. Since the early detection of cirrhosis is a clinical problem, achieving a sensitive, specific and predictive novel method based on biomarkers is an important task. Methods: Essential analysis, such as gene ontology (GO) enrichment and protein-protein interactions (PPI) was undergone EXPASy, STRING Database and DAVID Bioinformatics Resources query. Results: Based on GO analysis, most of proteins are located in the endoplasmic reticulum lumen, intracellular organelle lumen, membrane-enclosed lumen, and extracellular region. The relevant molecular functions are actin binding, metal ion binding, cation binding and ion binding. Cell adhesion, biological adhesion, cellular amino acid derivative, metabolic process and homeostatic process are the related processes. Protein-protein interaction network analysis introduced five proteins (fibroblast growth factor receptor 4, tropomyosin 4, tropomyosin 2 (beta), lectin, Lectin galactoside-binding soluble 3 binding protein and apolipoprotein A-I) as hub and bottleneck proteins. Conclusion: Our result indicates that regulation of lipid metabolism and cell survival are important biological processes involved in cirrhosis disease. More investigation of above mentioned proteins will provide a better understanding of cirrhosis disease. PMID:27099671

  20. An antifungal protein from Ginkgo biloba binds actin and can trigger cell death.

    PubMed

    Gao, Ningning; Wadhwani, Parvesh; Mühlhäuser, Philipp; Liu, Qiong; Riemann, Michael; Ulrich, Anne S; Nick, Peter

    2016-07-01

    Ginkbilobin is a short antifungal protein that had been purified and cloned from the seeds of the living fossil Ginkgo biloba. Homologues of this protein can be detected in all seed plants and the heterosporic fern Selaginella and are conserved with respect to domain structures, peptide motifs, and specific cysteine signatures. To get insight into the cellular functions of these conserved motifs, we expressed green fluorescent protein fusions of full-length and truncated ginkbilobin in tobacco BY-2 cells. We show that the signal peptide confers efficient secretion of ginkbilobin. When this signal peptide is either cleaved or masked, ginkbilobin binds and visualizes the actin cytoskeleton. This actin-binding activity of ginkbilobin is mediated by a specific subdomain just downstream of the signal peptide, and this subdomain can also coassemble with actin in vitro. Upon stable overexpression of this domain, we observe a specific delay in premitotic nuclear positioning indicative of a reduced dynamicity of actin. To elucidate the cellular response to the binding of this subdomain to actin, we use chemical engineering based on synthetic peptides comprising different parts of the actin-binding subdomain conjugated with the cell-penetrating peptide BP100 and with rhodamine B as a fluorescent reporter. Binding of this synthetic construct to actin efficiently induces programmed cell death. We discuss these findings in terms of a working model, where ginkbilobin can activate actin-dependent cell death. PMID:26315821

  1. A Novel Graphene Oxide-Based Protein Interaction Measurement Using Atomic Force Microscopy.

    PubMed

    Han, Sung-Woong; Morita, Kyohei; Adachi, Taiji

    2015-02-01

    Graphene oxide (GO) is a promising material for biological applications because of its excellent physical/chemical properties such as aqueous processability, amphiphilicity, and surface functionalizability. Here we introduce a new biological application of GO, a novel GO-based technique for probing protein interactions using atomic force microscopy (AFM). GO sheets were intercalated between the protein-modified AFM probe and the polymer substrate in order to reduce the non-specific adhesion force observed during single-molecule force spectroscopy (SMFS). In this study, we used SMFS to probe the interaction of the actin filament and actin-related protein 2/3 complex (Arp2/3), an actin-binding protein. Our results confirm that the GO sheet reduces nonspecific adhesion of the probe to the substrate. Using the GO-based technique, we succeeded in estimating the dissociation constant of the actin filament-binding protein interaction. PMID:26353630

  2. Development and Application of a High Throughput Protein Unfolding Kinetic Assay

    PubMed Central

    Wang, Qiang; Waterhouse, Nicklas; Feyijinmi, Olusegun; Dominguez, Matthew J.; Martinez, Lisa M.; Sharp, Zoey; Service, Rachel; Bothe, Jameson R.; Stollar, Elliott J.

    2016-01-01

    The kinetics of folding and unfolding underlie protein stability and quantification of these rates provides important insights into the folding process. Here, we present a simple high throughput protein unfolding kinetic assay using a plate reader that is applicable to the studies of the majority of 2-state folding proteins. We validate the assay by measuring kinetic unfolding data for the SH3 (Src Homology 3) domain from Actin Binding Protein 1 (AbpSH3) and its stabilized mutants. The results of our approach are in excellent agreement with published values. We further combine our kinetic assay with a plate reader equilibrium assay, to obtain indirect estimates of folding rates and use these approaches to characterize an AbpSH3-peptide hybrid. Our high throughput protein unfolding kinetic assays allow accurate screening of libraries of mutants by providing both kinetic and equilibrium measurements and provide a means for in-depth ϕ-value analyses. PMID:26745729

  3. Evolutionary and functional diversity of coronin proteins.

    PubMed

    Xavier, Charles-Peter; Eichinger, Ludwig; Fernandez, M Pilar; Morgan, Reginald O; Clemen, Christoph S

    2008-01-01

    This chapter discusses various aspects of coronin phylogeny, structure and function that are of specific interest. Two subfamilies of ancient coronins of unicellular pathogens such as Entamoeba, Trypanosoma, Leishmania and Acanthamoeba as well as of Plasmodium, Babesia, and Trichomonas are presented in the first two sections. Their coronins generally bind to F-actin and apparently are involved in proliferation, locomotion and phagocytosis. However, there are so far no studies addressing a putative role of coronin in the virulence of these pathogens. The following section delineates genetic anomalies like the chimeric coronin-fusion products with pelckstrin homology and gelsolin domains that are found in amoeba. Moreover, most nonvertebrate metazoa appear to encode CRN8, CRN9 and CRN7 representatives (for these coronin symbols see Chapter 2), but in e.g., Drosophila melanogaster and Caenorhabditis elegans a CRN9 is missing. The forth section deals with the evolutionary expansion of vertebrate coronins. Experimental data on the F-actin binding CRN2 of Xenopus (Xcoronin) including a Cdc42/Rac interactive binding (CRIB) motif that is also present in other members of the coronin protein family are discussed. Xenopus laevis represents a case for the expansion of the seven vertebrate coronins due to tetraploidization events. Other examples for a change in the number of coronin paralogs are zebrafish and birds, but (coronin) gene duplication events also occurred in unicellular protozoa. The fifth section of this chapter briefly summarizes three different cellular processes in which CRN4/CORO1A is involved, namely actin-binding, superoxide generation and Ca(2+)-signaling and refers to the largely unexplored mammalian coronins CRN5/CORO2A and CRN6/CORO2B, the latter binding to vinculin. The final section discusses how, by unveiling the aspects of coronin function in organisms reported so far, one can trace a remarkable evolution and diversity in their individual roles

  4. Structural characterization of a capping protein interaction motif defines a family of actin filament regulators

    PubMed Central

    Hernandez-Valladares, Maria; Kim, Taekyung; Kannan, Balakrishnan; Tung, Alvin; Aguda, Adeleke H; Larsson, Mårten; Cooper, John A; Robinson, Robert C

    2011-01-01

    Capping protein (CP) regulates actin dynamics by binding the barbed ends of actin filaments. Removal of CP may be one means to harness actin polymerization for processes such as cell movement and endocytosis. Here we structurally and biochemically investigated a CP interaction (CPI) motif present in the otherwise unrelated proteins CARMIL and CD2AP. The CPI motif wraps around the stalk of the mushroom-shaped CP at a site distant from the actin-binding interface, which lies on the top of the mushroom cap. We propose that the CPI motif may act as an allosteric modulator, restricting CP to a low-affinity, filament-binding conformation. Structure-based sequence alignments extend the CPI motif–containing family to include CIN85, CKIP-1, CapZIP and a relatively uncharacterized protein, WASHCAP (FAM21). Peptides comprising these CPI motifs are able to inhibit CP and to uncap CP-bound actin filaments. PMID:20357771

  5. The Gas2 family protein Pigs is a microtubule +TIP that affects cytoskeleton organisation

    PubMed Central

    Girdler, Gemma C.; Applewhite, Derek A.; Perry, Wick M. G.; Rogers, Stephen L.; Röper, Katja

    2016-01-01

    ABSTRACT Coordination between different cytoskeletal systems is crucial for many cell biological functions, including cell migration and mitosis, and also plays an important role during tissue morphogenesis. Proteins of the class of cytoskeletal crosslinkers, or cytolinkers, have the ability to interact with more than one cytoskeletal system at a time and are prime candidates to mediate any coordination. One such class comprises the Gas2-like proteins, combining a conserved calponin-homology-type actin-binding domain and a Gas2 domain predicted to bind microtubules (MTs). This domain combination is also found in spectraplakins, huge cytolinkers that play important roles in many tissues in both invertebrates and vertebrates. Here, we dissect the ability of the single Drosophila Gas2-like protein Pigs to interact with both actin and MT cytoskeletons, both in vitro and in vivo, and illustrate complex regulatory interactions that determine the localisation of Pigs to and its effects on the cytoskeleton. PMID:26585311

  6. The regulation and function of the striated muscle activator of rho signaling (STARS) protein

    PubMed Central

    Wallace, Marita A.; Lamon, Séverine; Russell, Aaron P.

    2012-01-01

    Healthy living throughout the lifespan requires continual growth and repair of cardiac, smooth, and skeletal muscle. To effectively maintain these processes muscle cells detect extracellular stress signals and efficiently transmit them to activate appropriate intracellular transcriptional programs. The striated muscle activator of Rho signaling (STARS) protein, also known as Myocyte Stress-1 (MS1) protein and Actin-binding Rho-activating protein (ABRA) is highly enriched in cardiac, skeletal, and smooth muscle. STARS binds actin, co-localizes to the sarcomere and is able to stabilize the actin cytoskeleton. By regulating actin polymerization, STARS also controls an intracellular signaling cascade that stimulates the serum response factor (SRF) transcriptional pathway; a pathway controlling genes involved in muscle cell proliferation, differentiation, and growth. Understanding the activation, transcriptional control and biological roles of STARS in cardiac, smooth, and skeletal muscle, will improve our understanding of physiological and pathophysiological muscle development and function. PMID:23248604

  7. Binding assay and preliminary X-ray crystallographic analysis of ACTIBIND, a protein with anticarcinogenic and antiangiogenic activities

    SciTech Connect

    Leeuw, Marina de; Roiz, Levava; Smirnoff, Patricia; Schwartz, Betty; Shoseyov, Oded; Almog, Orna

    2007-08-01

    Native ACTIBIND was successfully crystallized and it was shown that the interaction between ACTIBIND and actin is in a molar ratio of 1:2, with a binding constant of 16.17 × 10{sup 4} M{sup −1}. ACTIBIND is a T2 RNase extracellular glycoprotein produced by the mould Aspergillus niger B1 (CMI CC 324626) that possesses anticarcinogenic and antiangiogenic activities. ACTIBIND was found to be an actin-binding protein that interacts with rabbit muscle actin in a 1:2 molar ratio (ACTIBIND:actin) with a binding constant of 16.17 × 10{sup 4} M{sup −1}. Autoclave-treated ACTIBIND (EI-ACTIBIND) lost its RNase activity, but its actin-binding ability was conserved. ACTIBIND crystals were grown using 20% PEG 3350, 0.2 M ammonium dihydrogen phosphate solution at room temperature (293 K). One to four single crystals appeared in each droplet within a few days and grew to approximate dimensions of 0.5 × 0.5 × 0.5 mm after about two weeks. Diffraction studies of these crystals at low temperature (100 K) indicated that they belong to the P3{sub 1}21 space group, with unit-cell parameters a = 78, b = 78, c = 104 Å.

  8. Binding assay and preliminary X-ray crystallographic analysis of ACTIBIND, a protein with anticarcinogenic and antiangiogenic activities.

    PubMed

    de Leeuw, Marina; Roiz, Levava; Smirnoff, Patricia; Schwartz, Betty; Shoseyov, Oded; Almog, Orna

    2007-08-01

    ACTIBIND is a T2 RNase extracellular glycoprotein produced by the mould Aspergillus niger B1 (CMI CC 324626) that possesses anticarcinogenic and antiangiogenic activities. ACTIBIND was found to be an actin-binding protein that interacts with rabbit muscle actin in a 1:2 molar ratio (ACTIBIND:actin) with a binding constant of 16.17 x 10(4) M(-1). Autoclave-treated ACTIBIND (EI-ACTIBIND) lost its RNase activity, but its actin-binding ability was conserved. ACTIBIND crystals were grown using 20% PEG 3350, 0.2 M ammonium dihydrogen phosphate solution at room temperature (293 K). One to four single crystals appeared in each droplet within a few days and grew to approximate dimensions of 0.5 x 0.5 x 0.5 mm after about two weeks. Diffraction studies of these crystals at low temperature (100 K) indicated that they belong to the P3(1)21 space group, with unit-cell parameters a = 78, b = 78, c = 104 A. PMID:17671376

  9. Binding assay and preliminary X-ray crystallographic analysis of ACTIBIND, a protein with anticarcinogenic and antiangiogenic activities

    PubMed Central

    de Leeuw, Marina; Roiz, Levava; Smirnoff, Patricia; Schwartz, Betty; Shoseyov, Oded; Almog, Orna

    2007-01-01

    ACTIBIND is a T2 RNase extracellular glycoprotein produced by the mould Aspergillus niger B1 (CMI CC 324626) that possesses anticarcinogenic and antiangiogenic activities. ACTIBIND was found to be an actin-binding protein that interacts with rabbit muscle actin in a 1:2 molar ratio (ACTIBIND:actin) with a binding constant of 16.17 × 104  M −1. Autoclave-treated ACTIBIND (EI-ACTIBIND) lost its RNase activity, but its actin-binding ability was conserved. ACTIBIND crystals were grown using 20% PEG 3350, 0.2 M ammonium dihydrogen phosphate solution at room temperature (293 K). One to four single crystals appeared in each droplet within a few days and grew to approximate dimensions of 0.5 × 0.5 × 0.5 mm after about two weeks. Diffraction studies of these crystals at low temperature (100 K) indicated that they belong to the P3121 space group, with unit-cell parameters a = 78, b = 78, c = 104 Å. PMID:17671376

  10. The small heat shock-related protein, HSP20, is a cAMP-dependent protein kinase substrate that is involved in airway smooth muscle relaxation

    PubMed Central

    Komalavilas, Padmini; Penn, Raymond B.; Flynn, Charles R.; Thresher, Jeffrey; Lopes, Luciana B.; Furnish, Elizabeth J.; Guo, Manhong; Pallero, Manuel A.; Murphy-Ullrich, Joanne E.; Brophy, Colleen M.

    2009-01-01

    Activation of the cAMP/cAMP-dependent PKA pathway leads to relaxation of airway smooth muscle (ASM). The purpose of this study was to examine the role of the small heat shock-related protein HSP20 in mediating PKA-dependent ASM relaxation. Human ASM cells were engineered to constitutively express a green fluorescent protein-PKA inhibitory fusion protein (PKI-GFP) or GFP alone. Activation of the cAMP-dependent signaling pathways by isoproterenol (ISO) or forskolin led to increases in the phosphorylation of HSP20 in GFP but not PKI-GFP cells. Forskolin treatment in GFP but not PKI-GFP cells led to a loss of central actin stress fibers and decreases in the number of focal adhesion complexes. This loss of stress fibers was associated with dephosphorylation of the actin-depolymerizing protein cofilin in GFP but not PKI-GFP cells. To confirm that phosphorylated HSP20 plays a role in PKA-induced ASM relaxation, intact strips of bovine ASM were precontracted with serotonin followed by ISO. Activation of the PKA pathway led to relaxation of bovine ASM, which was associated with phosphorylation of HSP20 and dephosphorylation of cofilin. Finally, treatment with phosphopeptide mimetics of HSP20 possessing a protein transduction domain partially relaxed precontracted bovine ASM strips. In summary, ISO-induced phosphorylation of HSP20 or synthetic phosphopeptide analogs of HSP20 decreases phosphorylation of cofilin and disrupts actin in ASM, suggesting that one possible mechanism by which HSP20 mediates ASM relaxation is via regulation of actin filament dynamics. PMID:17993590

  11. PFA fixation enables artifact-free super-resolution imaging of the actin cytoskeleton and associated proteins.

    PubMed

    Leyton-Puig, Daniela; Kedziora, Katarzyna M; Isogai, Tadamoto; van den Broek, Bram; Jalink, Kees; Innocenti, Metello

    2016-01-01

    Super-resolution microscopy (SRM) allows precise localization of proteins in cellular organelles and structures, including the actin cytoskeleton. Yet sample preparation protocols for SRM are rather anecdotal and still being optimized. Thus, SRM-based imaging of the actin cytoskeleton and associated proteins often remains challenging and poorly reproducible. Here, we show that proper paraformaldehyde (PFA)-based sample preparation preserves the architecture of the actin cytoskeleton almost as faithfully as gold-standard glutaraldehyde fixation. We show that this fixation is essential for proper immuno-based localization of actin-binding and actin-regulatory proteins involved in the formation of lamellipodia and ruffles, such as mDia1, WAVE2 and clathrin heavy chain, and provide detailed guidelines for the execution of our method. In summary, proper PFA-based sample preparation increases the multi-color possibilities and the reproducibility of SRM of the actin cytoskeleton and its associated proteins. PMID:27378434

  12. PFA fixation enables artifact-free super-resolution imaging of the actin cytoskeleton and associated proteins

    PubMed Central

    Leyton-Puig, Daniela; Kedziora, Katarzyna M.; Isogai, Tadamoto; van den Broek, Bram; Jalink, Kees

    2016-01-01

    ABSTRACT Super-resolution microscopy (SRM) allows precise localization of proteins in cellular organelles and structures, including the actin cytoskeleton. Yet sample preparation protocols for SRM are rather anecdotal and still being optimized. Thus, SRM-based imaging of the actin cytoskeleton and associated proteins often remains challenging and poorly reproducible. Here, we show that proper paraformaldehyde (PFA)-based sample preparation preserves the architecture of the actin cytoskeleton almost as faithfully as gold-standard glutaraldehyde fixation. We show that this fixation is essential for proper immuno-based localization of actin-binding and actin-regulatory proteins involved in the formation of lamellipodia and ruffles, such as mDia1, WAVE2 and clathrin heavy chain, and provide detailed guidelines for the execution of our method. In summary, proper PFA-based sample preparation increases the multi-color possibilities and the reproducibility of SRM of the actin cytoskeleton and its associated proteins. PMID:27378434

  13. Proteomic analysis of ACTN4-interacting proteins reveals it's a putative involvement in mRNA metabolism

    SciTech Connect

    Khotin, Mikhail; Turoverova, Lidia; Aksenova, Vasilisa; Department of Genetics, St. Petersburg State University, Universitetskaya nab., 7 Barlev, Nikolai; Department of Biochemistry, University of Leicester, Lancaster Road, Leicester LE1 9HN ; Borutinskaite, Veronika Viktorija; Department of Developmental Biology, Institute of Biochemistry, LT-08662 Vilnius ; Vener, Alexander; Bajenova, Olga; Pinaev, George P.; Tentler, Dmitri

    2010-06-25

    Alpha-actinin 4 (ACTN4) is an actin-binding protein. In the cytoplasm, ACTN4 participates in structural organisation of the cytoskeleton via cross-linking of actin filaments. Nuclear localisation of ACTN4 has also been reported, but no clear role in the nucleus has been established. In this report, we describe the identification of proteins associated with ACTN4 in the nucleus. A combination of two-dimensional gel electrophoresis (2D-GE) and MALDI-TOF mass-spectrometry revealed a large number of ACTN4-bound proteins that are involved in various aspects of mRNA processing and transport. The association of ACTN4 with different ribonucleoproteins suggests that a major function of nuclear ACTN4 may be regulation of mRNA metabolism and signaling.

  14. MICAL-Family Proteins: Complex Regulators of the Actin Cytoskeleton

    PubMed Central

    Giridharan, Sai Srinivas Panapakkam

    2014-01-01

    Abstract Significance: The molecules interacting with CasL (MICAL) family members participate in a multitude of activities, including axonal growth cone repulsion, membrane trafficking, apoptosis, and bristle development in flies. An interesting feature of MICAL proteins is the presence of an N-terminal flavo-mono-oxygenase domain. This mono-oxygenase domain generates redox potential with which MICALs can either oxidize proteins or produce reactive oxygen species (ROS). Actin is one such protein that is affected by MICAL function, leading to dramatic cytoskeletal rearrangements. This review describes the MICAL-family members, and discusses their mechanisms of actin-binding and regulation of actin cytoskeleton organization. Recent Advances: Recent studies show that MICALs directly induce oxidation of actin molecules, leading to actin depolymerization. ROS production by MICALs also causes oxidation of collapsin response mediator protein-2, a microtubule assembly promoter, which subsequently undergoes phosphorylation. Critical Issues: MICAL proteins oxidize proteins through two mechanisms: either directly by oxidizing methionine residues or indirectly via the production of ROS. It remains unclear whether MICAL proteins employ both mechanisms or whether the activity of MICAL-family proteins might vary with different substrates. Future Directions: The identification of additional substrates oxidized by MICAL will shed new light on MICAL protein function. Additional directions include expanding studies toward the MICAL-like homologs that lack flavin adenine dinucleotide domains and oxidation activity. Antioxid. Redox Signal. 20, 2059–2073. PMID:23834433

  15. Chromaffin granule membrane-F-actin interactions and spectrin-like protein of subcellular organelles: a possible relationship.

    PubMed

    Aunis, D; Perrin, D

    1984-06-01

    The membrane of chromaffin granule, the secretory vesicle of adrenal medullary cells storing catecholamines, enkephalins, and many other components, interacts with F-actin. Using low shear falling ball viscometry to estimate actin binding to membranes, we demonstrated that mitochondrial and plasma membranes from chromaffin cells also provoked large increases in viscosity of F-actin solutions. Mitochondrial membranes also had the capacity to cause complete gelation of F-actin. In addition, vasopressin-containing granules from neurohypophysial tissue were shown to bind F-actin and to increase the viscosity of F-actin solutions. Using an antibody directed against human erythrocyte spectrin, it was found that a spectrin-like protein was associated with secretory granule membrane, mitochondrial membrane, and plasma membrane. The chromaffin granule membrane-associated spectrin-like protein faces the cytoplasmic side, is composed of two subunits (240 kD and 235kD ), the alpha-subunit (240 kD, pHi5 .5) being recognized by the antibody. Nonionic detergents such as Triton X-100 or Nonidet P40 failed to release fully active spectrin-like protein. In contrast, Kyro EOB , a different nonionic detergent, was found to release spectrin-like protein while keeping intact F-actin binding capacity, at least below 0.5% Kyro EOB concentration. Chromaffin cells in culture were stained with antispectrin antibody, showing the presence of spectrin-like protein in the cell periphery close to the cell membrane but also in the cytoplasm. We conclude that in living cells the interaction of F-actin with chromaffin granule membrane spectrin observed in vitro is important in controlling the potential function of secretory vesicles. PMID:6374036

  16. Cytoskeletal proteins in gastric H/sup +/ secretion: cAMP dependent phosphorylation, immunolocalization, and protein blotting

    SciTech Connect

    Cuppoletti, J.; Sachs, G.; Malinowska, D.H.

    1986-05-01

    The rabbit gastric parietal cell is an excellent model for the study of regulation of secretion and the role of cytoskeleton in secretion. Changes in morphology (appearance of expanded secretory canaliculi lined with microvilli) accompany H/sup +/ secretion stimulated by histamine (cAMP mediated). Parietal cells contain immunoreactive tubulin and are highly enriched in F-actin at secretory canaliculi, detected with fluorescently labelled phallacidin. They have previously shown increased protein phosphorylation in histamine-stimulated purified parietal cells concommitant with increases in H/sup +/ secretion. They report here possible functions of the phosphoproteins. Four of these proteins of apparent size on SDS PAGE of 24, 30, 48 and 130 Kd were membrane associated. /sup 125/I-actin binding to three proteins (24, 30 and 48 Kd) was shown using overlays. A 130 Kd protein reacted with anti-vinculin monoclonal antibody on immunoblots, and was immunolocalized at secretory canaliculi. As a working hypothesis, parietal cells possess membrane-associated proteins which change their state of phosphorylation upon stimulation of H/sup +/. These proteins may be cytoskeletal elements involved in regulation of H/sup +/ secretion. The 130 Kd vinculin-like protein may serve a microfilament-membrane linking role.

  17. 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

  18. Cytoplasmic protein methylation is essential for neural crest migration

    PubMed Central

    Vermillion, Katie L.; Lidberg, Kevin A.

    2014-01-01

    As they initiate migration in vertebrate embryos, neural crest cells are enriched for methylation cycle enzymes, including S-adenosylhomocysteine hydrolase (SAHH), the only known enzyme to hydrolyze the feedback inhibitor of trans-methylation reactions. The importance of methylation in neural crest migration is unknown. Here, we show that SAHH is required for emigration of polarized neural crest cells, indicating that methylation is essential for neural crest migration. Although nuclear histone methylation regulates neural crest gene expression, SAHH and lysine-methylated proteins are abundant in the cytoplasm of migratory neural crest cells. Proteomic profiling of cytoplasmic, lysine-methylated proteins from migratory neural crest cells identified 182 proteins, several of which are cytoskeleton related. A methylation-resistant form of one of these proteins, the actin-binding protein elongation factor 1 alpha 1 (EF1α1), blocks neural crest migration. Altogether, these data reveal a novel and essential role for post-translational nonhistone protein methylation during neural crest migration and define a previously unknown requirement for EF1α1 methylation in migration. PMID:24379414

  19. Facioscapulohumeral muscular dystrophy region gene 1 (FRG1) is a dynamic RNA-associated and actin bundling protein

    PubMed Central

    Jessica Sun, Chia-Yun; van Koningsbruggen, Silvana; Long, Steven W.; Straasheijm, Kirsten; Klooster, Rinse; Jones, Takako I.; Bellini, Michel; Levesque, Lyne; Brieher, William M.; van der Maarel, Silvère M.; Jones, Peter L.

    2011-01-01

    Facioscapulohumeral muscular dystrophy (FSHD) region gene 1 (FRG1) is a dynamic nuclear and cytoplasmic protein that, in skeletal muscle, shows an additional localization to the sarcomere. Maintaining appropriate levels of FRG1 protein is critical for the muscle and vascular development in the vertebrate, however its precise molecular function is unknown. This study investigates the molecular functions of human FRG1 along with mouse and Xenopus frg1 using molecular, biochemical and cell-biological approach to provide further insight into its roles in vertebrate development. The nuclear fraction of the endogenous FRG1 is localized in nucleoli, Cajal bodies, and actively transcribed chromatin; however, contrary to overexpressed FRG1, the endogenous FRG1 is not associated with nuclear speckles. We characterize the nuclear and nucleolar import of FRG1, the potential role for phosphorylation, and its interaction with the importin karyophernα2 (KPNA2). Consistent with a role in RNA biogenesis, human FRG1 is associated with mRNA in vivo and in vitro and interacts directly with TAP, the major mRNA export receptor, and is a dynamic nuclear-cytoplasmic shuttling protein supporting a function for FRG1 in mRNA transport. Biochemically, we characterize FRG1 actin binding activity and show that the cytoplasmic pool of FRG1 is dependent on an intact actin cytoskeleton for its localization. These data provide the first biochemical activities - actin binding and RNA binding - for human FRG1 and the characterizations of the endogenous human FRG1, together indicating FRG1 is involved in multiple aspects of RNA biogenesis including mRNA transport and potentially cytoplasmic mRNA localization. PMID:21699900

  20. Screening Molecular Chaperones Similar to Small Heat Shock Proteins in Schizosaccharomyces pombe

    PubMed Central

    Han, Jiyoung; Kim, Kanghwa

    2015-01-01

    To screen molecular chaperones similar to small heat shock proteins (sHsps), but without α-crystalline domain, heat-stable proteins from Schizosaccharomyces pombe were analyzed by 2-dimensional electrophoresis and matrix assisted laser desorption/ionization time-of-flight mass spectrometry. Sixteen proteins were identified, and four recombinant proteins, including cofilin, NTF2, pyridoxin biosynthesis protein (Snz1) and Wos2 that has an α-crystalline domain, were purified. Among these proteins, only Snz1 showed the anti-aggregation activity against thermal denaturation of citrate synthase. However, pre-heating of NTF2 and Wos2 at 70℃ for 30 min, efficiently prevented thermal aggregation of citrate synthase. These results indicate that Snz1 and NTF2 possess molecular chaperone activity similar to sHsps, even though there is no α-crystalline domain in their sequences. PMID:26539043

  1. The Genetic Engineering of Motor Proteins

    NASA Astrophysics Data System (ADS)

    Hartz, Rachael M.

    Molecular motors are a remarkable feature within living organisms that are responsible for directional mechanical motion, which is driven by adenosine triphosphate (ATP) hydrolysis. Actin-binding molecular motors are of specific interest in the field of nanotechnology as filamentous actin is capable of carrying cargo, such as quantum dots, while it is translocated along a motor coated surface. The binding regions of motor proteins, which are known to interact with actin, such as Myosin, have been thoroughly examined and identified. Rapid genetic engineering of the ATP-hydrolyzing enzyme, adenosine kinase, to incorporate these binding regions is possible through the use of site- directed mutagenesis. The sequences, which were mutated into the ADK wt gene, were incorporated in an unstructured loop region. During the phosphate transfer, the mutants switch between open and closed conformational states. The binding affinity of the sequences to the actin is altered during this conformational switch, thus causing the motor to move along actin filament. The ADK mutants and their interaction with filamentous actin was monitored by an in vitro motility assay. Two different mutants of ADK were found to have retained enzymatic functionality after the mutagenesis as well as function as actin-based motor proteins.

  2. 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...

  3. 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)

  4. Analysis of the Conformation and Function of the Plasmodium falciparum Merozoite Proteins MTRAP and PTRAMP

    PubMed Central

    Uchime, Onyinyechukwu; Herrera, Raul; Reiter, Karine; Kotova, Svetlana; Shimp, Richard L.; Miura, Kazutoyo; Jones, Dominique; Lebowitz, Jacob; Ambroggio, Xavier; Hurt, Darrell E.; Jin, Albert J.; Long, Carole; Miller, Louis H.

    2012-01-01

    Thrombospondin repeat (TSR)-like domains are structures involved with cell adhesion. Plasmodium falciparum proteins containing TSR domains play crucial roles in parasite development. In particular, the preerythrocytic P. falciparum circumsporozoite protein is involved in hepatocyte invasion. The importance of these domains in two other malaria proteins, the merozoite-specific thrombospondin-related anonymous protein (MTRAP) and the thrombospondin-related apical membrane protein (PTRAMP), were assessed using near-full-length recombinant proteins composed of the extracellular domains produced in Escherichia coli. MTRAP is thought to be released from invasive organelles identified as micronemes during merozoite invasion to mediate motility and host cell invasion through an interaction with aldolase, an actin binding protein involved in the moving junction. PTRAMP function remains unknown. In this study, the conformation of recombinant MTRAP (rMTRAP) appeared to be a highly extended protein (2 nm by 33 nm, width by length, respectively), whereas rPTRAMP had a less extended structure. Using an erythrocyte binding assay, rMTRAP but not rPTRAMP bound human erythrocytes; rMTRAP binding was mediated through the TSR domain. MTRAP- and in general PTRAMP-specific antibodies failed to inhibit P. falciparum development in vitro. Altogether, MTRAP is a highly extended bifunctional protein that binds to an erythrocyte receptor and the merozoite motor. PMID:22467743

  5. Nesprin-3, a novel outer nuclear membrane protein, associates with the cytoskeletal linker protein plectin

    PubMed Central

    Wilhelmsen, Kevin; Litjens, Sandy H.M.; Kuikman, Ingrid; Tshimbalanga, Ntambua; Janssen, Hans; van den Bout, Iman; Raymond, Karine; Sonnenberg, Arnoud

    2005-01-01

    Despite their importance in cell biology, the mechanisms that maintain the nucleus in its proper position in the cell are not well understood. This is primarily the result of an incomplete knowledge of the proteins in the outer nuclear membrane (ONM) that are able to associate with the different cytoskeletal systems. Two related ONM proteins, nuclear envelope spectrin repeat (nesprin)–1 and –2, are known to make direct connections with the actin cytoskeleton through their NH2-terminal actin-binding domain (ABD). We have now isolated a third member of the nesprin family that lacks an ABD and instead binds to the plakin family member plectin, which can associate with the intermediate filament (IF) system. Overexpression of nesprin-3 results in a dramatic recruitment of plectin to the nuclear perimeter, which is where these two molecules are colocalized with both keratin-6 and -14. Importantly, plectin binds to the integrin α6β4 at the cell surface and to nesprin-3 at the ONM in keratinocytes, suggesting that there is a continuous connection between the nucleus and the extracellular matrix through the IF cytoskeleton. PMID:16330710

  6. Protein Motions and Folding Investigated by NMR Spectroscopy

    NASA Astrophysics Data System (ADS)

    Palmer, Arthur

    2002-03-01

    NMR spin relaxation spectroscopy is a powerful experimental approach for globally characterizing conformational dynamics of proteins in solution. Laboratory frame relaxation measurements are sensitive to overall rotational diffusion and internal motions on picosecond-nanosecond time scales, while rotating frame relaxation measurements are sensitive to chemical exchange processes on microsecond-millisecond time scales. The former approach is illustrated by ^15N laboratory-frame relaxation experiments as a function of temperature for the helical subdomain HP36 of the F-actin-binding headpiece domain of chicken villin. The data are analyzed using the model-free formalism to characterize order parameters and effective correlation times for intramolecular motions of individual ^15N sites. The latter approach is illustrated by ^13C Carr-Purcell-Meiboom-Gill relaxation measurements for the de novo designed α_2D protein and by ^15N rotating-frame relaxation measurements for the peripheral subunit-binding domain (PSBD) from the dihydrolopoamide acetyltransferase component of the pyruvate dehydrogenase multienzyme complex from Bacillus stearothermophilus. These experiments are used to determine the folding and unfolding kinetic rate constants for the two proteins. The results for HP36, α_2D, and PSBD illustrate the capability of current NMR methods for characterizing dynamic processes on multiple time scales in proteins.

  7. Sequence and comparative genomic analysis of actin-related proteins.

    PubMed

    Muller, Jean; Oma, Yukako; Vallar, Laurent; Friederich, Evelyne; Poch, Olivier; Winsor, Barbara

    2005-12-01

    Actin-related proteins (ARPs) are key players in cytoskeleton activities and nuclear functions. Two complexes, ARP2/3 and ARP1/11, also known as dynactin, are implicated in actin dynamics and in microtubule-based trafficking, respectively. ARP4 to ARP9 are components of many chromatin-modulating complexes. Conventional actins and ARPs codefine a large family of homologous proteins, the actin superfamily, with a tertiary structure known as the actin fold. Because ARPs and actin share high sequence conservation, clear family definition requires distinct features to easily and systematically identify each subfamily. In this study we performed an in depth sequence and comparative genomic analysis of ARP subfamilies. A high-quality multiple alignment of approximately 700 complete protein sequences homologous to actin, including 148 ARP sequences, allowed us to extend the ARP classification to new organisms. Sequence alignments revealed conserved residues, motifs, and inserted sequence signatures to define each ARP subfamily. These discriminative characteristics allowed us to develop ARPAnno (http://bips.u-strasbg.fr/ARPAnno), a new web server dedicated to the annotation of ARP sequences. Analyses of sequence conservation among actins and ARPs highlight part of the actin fold and suggest interactions between ARPs and actin-binding proteins. Finally, analysis of ARP distribution across eukaryotic phyla emphasizes the central importance of nuclear ARPs, particularly the multifunctional ARP4. PMID:16195354

  8. Scapinin, the Protein Phosphatase 1 Binding Protein, Enhances Cell Spreading and Motility by Interacting with the Actin Cytoskeleton

    PubMed Central

    Sagara, Junji; Arata, Toshiaki; Taniguchi, Shunichiro

    2009-01-01

    Scapinin, also named phactr3, is an actin and protein phosphatase 1 (PP1) binding protein, which is expressed in the adult brain and some tumor cells. At present, the role(s) of scapinin in the brain and tumors are poorly understood. We show that the RPEL-repeat domain of scapinin, which is responsible for its direct interaction with actin, inhibits actin polymerization in vitro. Next, we established a Hela cell line, where scapinin expression was induced by tetracycline. In these cells, expression of scapinin stimulated cell spreading and motility. Scapinin was colocalized with actin at the edge of spreading cells. To explore the roles of the RPEL-repeat and PP1-binding domains, we expressed wild-type and mutant scapinins as fusion proteins with green fluorescence protein (GFP) in Cos7 cells. Expression of GFP-scapinin (wild type) also stimulated cell spreading, but mutation in the RPEL-repeat domain abolished both the actin binding and the cell spreading activity. PP1-binding deficient mutants strongly induced cell retraction. Long and branched cytoplasmic processes were developed during the cell retraction. These results suggest that scapinin enhances cell spreading and motility through direct interaction with actin and that PP1 plays a regulatory role in scapinin-induced morphological changes. PMID:19158953

  9. AMP-activated protein kinase induces actin cytoskeleton reorganization in epithelial cells

    SciTech Connect

    Miranda, Lisa; Carpentier, Sarah; Platek, Anna; Hussain, Nusrat; Gueuning, Marie-Agnes; Vertommen, Didier; Ozkan, Yurda; Sid, Brice; Hue, Louis; Courtoy, Pierre J.; Rider, Mark H.; Horman, Sandrine

    2010-06-04

    AMP-activated protein kinase (AMPK), a known regulator of cellular and systemic energy balance, is now recognized to control cell division, cell polarity and cell migration, all of which depend on the actin cytoskeleton. Here we report the effects of A769662, a pharmacological activator of AMPK, on cytoskeletal organization and signalling in epithelial Madin-Darby canine kidney (MDCK) cells. We show that AMPK activation induced shortening or radiation of stress fibers, uncoupling from paxillin and predominance of cortical F-actin. In parallel, Rho-kinase downstream targets, namely myosin regulatory light chain and cofilin, were phosphorylated. These effects resembled the morphological changes in MDCK cells exposed to hyperosmotic shock, which led to Ca{sup 2+}-dependent AMPK activation via calmodulin-dependent protein kinase kinase-{beta}(CaMKK{beta}), a known upstream kinase of AMPK. Indeed, hypertonicity-induced AMPK activation was markedly reduced by the STO-609 CaMKK{beta} inhibitor, as was the increase in MLC and cofilin phosphorylation. We suggest that AMPK links osmotic stress to the reorganization of the actin cytoskeleton.

  10. Identification of the secreted watery saliva proteins of the rice brown planthopper, Nilaparvata lugens (Stål) by transcriptome and Shotgun LC-MS/MS approach.

    PubMed

    Liu, Xiaoqing; Zhou, Hanyu; Zhao, Jing; Hua, Hongxia; He, Yueping

    2016-06-01

    The rice brown planthopper, Nilaparvata lugens (Stål), a major rice insect pest in Asia, is a vascular bundle-feeder that ejects gelling and watery saliva during the feeding process. Although major proteins in the salivary glands of N. lugens have been identified using 2D PAGE, very little is known about the secreted saliva of this insect. In this study, we identified the major proteins in the secreted watery saliva of N. lugens, via collecting from a sucrose diet that adult planthoppers had fed upon through a membrane of stretched parafilm, and using shotgun LC-MS/MS analysis with reference to transcriptome database of salivary glands of N. lugens. A total of 107 proteins were identified in the watery saliva of N. lugens, over 80% of which showed significant similarity to known proteins. When annotated by the Blast2GO suite, 29 proteins had catalytic activity and 24 proteins were binding proteins. The saliva enzymes included oxidoreductases, hydrolases, phosphatases, peptidases (proteases), kinases, transferases, and lyases. Binding proteins in N. lugens watery saliva included ATP-binding, lipophorin, calcium-binding, actin-binding and DNA-, RNA-, and chromatin-binding proteins. Other non-enzymatic proteins, such as ubiquitins, heat shock proteins, ribosomal proteins, and immunoglobulin proteins were also found in N. lugens watery saliva. This is the first study to identify, characterize and list the proteins in watery saliva of N. lugens, which might be involved in planthopper-rice interactions. PMID:27080912

  11. Phosphotyrosine-dependent targeting of mitogen-activated protein kinase in differentiated contractile vascular cells.

    PubMed

    Khalil, R A; Menice, C B; Wang, C L; Morgan, K G

    1995-06-01

    Tyrosine phosphorylation has been linked to plasmalemmal targeting of src homology-2-containing proteins, activation of mitogen-activated protein (MAP) kinase, nuclear signaling, and proliferation of cultured cells. Significant tyrosine phosphorylation and MAP kinase activities have also been reported in differentiated cells, but the signaling role of tyrosine-phosphorylated MAP kinase in these cells is unclear. The spatial and temporal relation between phosphotyrosine and MAP kinase immunoreactivity was quantified in differentiated contractile vascular smooth muscle cells by using digital imaging microscopy. An initial association of MAP kinase with the plasmalemma required upstream protein kinase C activity but occurred in a tyrosine phosphorylation-independent manner. Subsequent to membrane association, a delayed redistribution of MAP kinase, colocalizing with the actin-binding protein caldesmon, occurred in a tyrosine phosphorylation-dependent manner. The apparent association of MAP kinase with the contractile proteins coincided with contractile activation. Thus, tyrosine phosphorylation appears to target MAP kinase to cytoskeletal proteins in contractile vascular cells. This targeting mechanism may determine the specific destination and thereby the specialized function of MAP kinase in other phenotypes. PMID:7538916

  12. Redundant control of migration and adhesion by ERM proteins in vascular smooth muscle cells

    SciTech Connect

    Baeyens, Nicolas; Latrache, Iman; Yerna, Xavier; Noppe, Gauthier; Horman, Sandrine; Morel, Nicole

    2013-11-22

    Highlights: •The three ERM proteins are expressed in vascular smooth muscle cell. •ERM depletion inhibited PDGF-evoked migration redundantly. •ERM depletion increased cell adhesion redundantly. •ERM depletion did not affect PDGF-evoked Ca signal, Rac1 activation, proliferation. •ERM proteins control PDGF-induced migration by regulating adhesion. -- Abstract: Ezrin, radixin, and moesin possess a very similar structure with a C-terminal actin-binding domain and a N-terminal FERM interacting domain. They are known to be involved in cytoskeleton organization in several cell types but their function in vascular smooth muscle cells (VSMC) is still unknown. The aim of this study was to investigate the role of ERM proteins in cell migration induced by PDGF, a growth factor involved in pathophysiological processes like angiogenesis or atherosclerosis. We used primary cultured VSMC obtained from rat aorta, which express the three ERM proteins. Simultaneous depletion of the three ERM proteins with specific siRNAs abolished the effects of PDGF on cell architecture and migration and markedly increased cell adhesion and focal adhesion size, while these parameters were only slightly affected by depletion of ezrin, radixin or moesin alone. Rac1 activation, cell proliferation, and Ca{sup 2+} signal in response to PDGF were unaffected by ERM depletion. These results indicate that ERM proteins exert a redundant control on PDGF-induced VSMC migration by regulating focal adhesion turn-over and cell adhesion to substrate.

  13. An update on cell surface proteins containing extensin-motifs.

    PubMed

    Borassi, Cecilia; Sede, Ana R; Mecchia, Martin A; Salgado Salter, Juan D; Marzol, Eliana; Muschietti, Jorge P; Estevez, Jose M

    2016-01-01

    In recent years it has become clear that there are several molecular links that interconnect the plant cell surface continuum, which is highly important in many biological processes such as plant growth, development, and interaction with the environment. The plant cell surface continuum can be defined as the space that contains and interlinks the cell wall, plasma membrane and cytoskeleton compartments. In this review, we provide an updated view of cell surface proteins that include modular domains with an extensin (EXT)-motif followed by a cytoplasmic kinase-like domain, known as PERKs (for proline-rich extensin-like receptor kinases); with an EXT-motif and an actin binding domain, known as formins; and with extracellular hybrid-EXTs. We focus our attention on the EXT-motifs with the short sequence Ser-Pro(3-5), which is found in several different protein contexts within the same extracellular space, highlighting a putative conserved structural and functional role. A closer understanding of the dynamic regulation of plant cell surface continuum and its relationship with the downstream signalling cascade is a crucial forthcoming challenge. PMID:26475923

  14. The Cytoskeleton and Force Response Mechanisms

    NASA Technical Reports Server (NTRS)

    Allen, Philip Goodwin

    2003-01-01

    examined the roles of several actin filament disassembly factors in the global reduction of cellular actin filaments. The calcium regulated actin filament severing protein gelsolin is not necessary for the increased actin turnover, as cells derived from gelsolin null and wildtype mice still show a reduction in total actin filament content. Instead, our work suggests that the actin binding protein cofilin may be important for these changes in actin dynamics. Cofilin binds to and enhances the disassembly of actin filaments. Using immunological methods, we observe transient changes in the phosphorylation state of cofilin upon force application that suggests that cofilin may mediate actin filament turnover. Early after force application, cofilin is transiently dephosphorylated, activating its actin disassembly activity. Subsequently, we find a hyper-phosphorylation of cofilin, rendering it inactive. This reduction in cofilin activity may explain the stability of the force induced actin structuttes. In testing this hypothesis, we aimed to generate cells that express the constituitively active kinase (LIM-kinase) that phosphorylates cofilin. lnidial attempts in the cell lines used for the our previous studies proved unsuccessful. While we prepare this work for pubication, we are continuing to study other cell lines and tissue sources to determine whether they show a reduction in F-actin content after force application.

  15. Diffusion of GPI-anchored proteins is influenced by the activity of dynamic cortical actin

    PubMed Central

    Saha, Suvrajit; Lee, Il-Hyung; Polley, Anirban; Groves, Jay T.; Rao, Madan; Mayor, Satyajit

    2015-01-01

    Molecular diffusion at the surface of living cells is believed to be predominantly driven by thermal kicks. However, there is growing evidence that certain cell surface molecules are driven by the fluctuating dynamics of cortical cytoskeleton. Using fluorescence correlation spectroscopy, we measure the diffusion coefficient of a variety of cell surface molecules over a temperature range of 24–37°C. Exogenously incorporated fluorescent lipids with short acyl chains exhibit the expected increase of diffusion coefficient over this temperature range. In contrast, we find that GPI-anchored proteins exhibit temperature-independent diffusion over this range and revert to temperature-dependent diffusion on cell membrane blebs, in cells depleted of cholesterol, and upon acute perturbation of actin dynamics and myosin activity. A model transmembrane protein with a cytosolic actin-binding domain also exhibits the temperature-independent behavior, directly implicating the role of cortical actin. We show that diffusion of GPI-anchored proteins also becomes temperature dependent when the filamentous dynamic actin nucleator formin is inhibited. However, changes in cortical actin mesh size or perturbation of branched actin nucleator Arp2/3 do not affect this behavior. Thus cell surface diffusion of GPI-anchored proteins and transmembrane proteins that associate with actin is driven by active fluctuations of dynamic cortical actin filaments in addition to thermal fluctuations, consistent with expectations from an “active actin-membrane composite” cell surface. PMID:26378258

  16. Translocation of an 89-kDa periplasmic protein is associated with Holospora infection

    SciTech Connect

    Iwatani, Koichi; Dohra, Hideo; Lang, B. Franz; Burger, Gertraud; Hori, Manabu; Fujishima, Masahiro . E-mail: fujishim@yamaguchi-u.ac.jp

    2005-12-02

    The symbiotic bacterium Holospora obtusa infects the macronucleus of the ciliate Paramecium caudatum. After ingestion by its host, an infectious form of Holospora with an electron-translucent tip passes through the host digestive vacuole and penetrates the macronuclear envelope with this tip. To investigate the underlying molecular mechanism of this process, we raised a monoclonal antibody against the tip-specific 89-kDa protein, sequenced this partially, and identified the corresponding complete gene. The deduced protein sequence carries two actin-binding motifs. Indirect immunofluorescence microscopy shows that during escape from the host digestive vacuole, the 89-kDa proteins translocates from the inside to the outside of the tip. When the bacterium invades the macronucleus, the 89-kDa protein is left behind at the entry point of the nuclear envelope. Transmission electron microscopy shows the formation of fine fibrous structures that co-localize with the antibody-labeled regions of the bacterium. Our findings suggest that the 89-kDa protein plays a role in Holospora's escape from the host digestive vacuole, the migration through the host cytoplasm, and the invasion into the macronucleus.

  17. Plasmodium falciparum cysteine protease falcipain-2 cleaves erythrocyte membrane skeletal proteins at late stages of parasite development.

    PubMed

    Hanspal, Manjit; Dua, Meenakshi; Takakuwa, Yuichi; Chishti, Athar H; Mizuno, Akiko

    2002-08-01

    Plasmodium falciparum-derived cysteine protease falcipain-2 cleaves host erythrocyte hemoglobin at acidic pH and specific components of the membrane skeleton at neutral pH. Analysis of stage-specific expression of these 2 proteolytic activities of falcipain-2 shows that hemoglobin-hydrolyzing activity is maximum in early trophozoites and declines rapidly at late stages, whereas the membrane skeletal protein hydrolyzing activity is markedly increased at the late trophozoite and schizont stages. Among the erythrocyte membrane skeletal proteins, ankyrin and protein 4.1 are cleaved by native and recombinant falcipain-2 near their C-termini. To identify the precise peptide sequence at the hydrolysis site of protein 4.1, we used a recombinant construct of protein 4.1 as substrate followed by MALDI-MS analysis of the cleaved product. We show that falcipain-2-mediated cleavage of protein 4.1 occurs immediately after lysine 437, which lies within a region of the spectrin-actin-binding domain critical for erythrocyte membrane stability. A 16-mer peptide containing the cleavage site completely inhibited the enzyme activity and blocked falcipain-2-induced fragmentation of erythrocyte ghosts. Based on these results, we propose that falcipain-2 cleaves hemoglobin in the acidic food vacuole at the early trophozoite stage, whereas it cleaves specific components of the red cell skeleton at the late trophozoite and schizont stages. It is the proteolysis of skeletal proteins that causes membrane instability, which, in turn, facilitates parasite release in vivo. PMID:12130521

  18. HSPC280, a winged helix protein expressed in the subventricular zone of the developing ganglionic eminences, inhibits neuronal differentiation.

    PubMed

    Stylianopoulou, Electra; Kalamakis, Georgios; Pitsiani, Margarita; Fysekis, Ioannis; Ypsilantis, Petros; Simopoulos, Constantinos; Skavdis, George; Grigoriou, Maria E

    2016-02-01

    Winged helix proteins have critical roles in a variety of developmental processes. During a screening for genes expressed in the developing forebrain, we identified HSPC280, a non-typical winged helix protein, which shares similarity with a protein-protein interaction domain found in the proteins of the actin-binding Rho-activating protein family. In this work, we analyzed HSPC280 expression during mouse development as well as during neuronal differentiation of mouse Neuro2a cells. HSPC280 expression is tightly regulated; during mouse development, it was detected predominantly in the ganglionic eminences of the ventral telencephalon, from their appearance at E11.5 to P0, with the highest levels between E13.5 and E15.5, a period that correlates with the peak of neurogenesis in these structures. Comparative expression analysis of HSPC280 with Dlx2, cyclinD2 and Lhx6 revealed that, within the ganglionic eminences, HSPC280 was restricted in the proliferating cell population of the subventricular zone, in a pattern similar to that of cyclinD2. Finally, we showed that HSPC280 is a nuclear protein which, when overexpressed in Neuro2a cells, it inhibited neuronal differentiation in vitro, suggesting its involvement in the mechanisms controlling neural progenitor cells proliferation. PMID:26537243

  19. Metabolic and evolutionary origin of actin-binding polyketides from diverse organisms.

    PubMed

    Ueoka, Reiko; Uria, Agustinus R; Reiter, Silke; Mori, Tetsushi; Karbaum, Petra; Peters, Eike E; Helfrich, Eric J N; Morinaka, Brandon I; Gugger, Muriel; Takeyama, Haruko; Matsunaga, Shigeki; Piel, Jörn

    2015-09-01

    Actin-targeting macrolides comprise a large, structurally diverse group of cytotoxins isolated from remarkably dissimilar micro- and macroorganisms. In spite of their disparate origins and structures, many of these compounds bind actin at the same site and exhibit structural relationships reminiscent of modular, combinatorial drug libraries. Here we investigate biosynthesis and evolution of three compound groups: misakinolides, scytophycin-type compounds and luminaolides. For misakinolides from the sponge Theonella swinhoei WA, our data suggest production by an uncultivated 'Entotheonella' symbiont, further supporting the relevance of these bacteria as sources of bioactive polyketides and peptides in sponges. Insights into misakinolide biosynthesis permitted targeted genome mining for other members, providing a cyanobacterial luminaolide producer as the first cultivated source for this dimeric compound family. The data indicate that this polyketide family is bacteria-derived and that the unusual macrolide diversity is the result of combinatorial pathway modularity for some compounds and of convergent evolution for others. PMID:26236936

  20. Analysis of Cytoskeletal and Motility Proteins in the Sea Urchin Genome Assembly

    PubMed Central

    RL, Morris; MP, Hoffman; RA, Obar; SS, McCafferty; IR, Gibbons; AD, Leone; J, Cool; EL, Allgood; AM, Musante; KM, Judkins; BJ, Rossetti; AP, Rawson; DR, Burgess

    2007-01-01

    The sea urchin embryo is a classical model system for studying the role of the cytoskeleton in such events as fertilization, mitosis, cleavage, cell migration and gastrulation. We have conducted an analysis of gene models derived from the Strongylocentrotus purpuratus genome assembly and have gathered strong evidence for the existence of multiple gene families encoding cytoskeletal proteins and their regulators in sea urchin. While many cytoskeletal genes have been cloned from sea urchin with sequences already existing in public databases, genome analysis reveals a significantly higher degree of diversity within certain gene families. Furthermore, genes are described corresponding to homologs of cytoskeletal proteins not previously documented in sea urchins. To illustrate the varying degree of sequence diversity that exists within cytoskeletal gene families, we conducted an analysis of genes encoding actins, specific actin-binding proteins, myosins, tubulins, kinesins, dyneins, specific microtubule-associated proteins, and intermediate filaments. We conducted ontological analysis of select genes to better understand the relatedness of urchin cytoskeletal genes to those of other deuterostomes. We analyzed developmental expression (EST) data to confirm the existence of select gene models and to understand their differential expression during various stages of early development. PMID:17027957

  1. FHOD proteins in actin dynamics—a formin’ class of its own

    PubMed Central

    Bechtold, Meike; Schultz, Jörg; Bogdan, Sven

    2014-01-01

    Eukaryotic cells have evolved a variety of actin-binding proteins to regulate the architecture and the dynamics of the actin cytoskeleton in time and space. The Diaphanous-related formins (DRF) represent a diverse group of Rho-GTPase-regulated actin regulators that control a range of actin structures composed of tightly-bundled, unbranched actin filaments as found in stress fibers and in filopodia. Under resting conditions, DRFs are auto-inhibited by an intra-molecular interaction between the C-terminal and the N-terminal domains. The auto-inhibition is thought to be released by binding of an activated RhoGTPase to the N-terminal GTPase-binding domain (GBD). However, there is growing evidence for more sophisticated variations from this simplified linear activation model. In this review we focus on the formin homology domain-containing proteins (FHOD), an unconventional group of DRFs. Recent findings on the molecular control and cellular functions of FHOD proteins in vivo are discussed in the light of the phylogeny of FHOD proteins. PMID:25483300

  2. The focal adhesion protein PINCH-1 associates with EPLIN at integrin adhesion sites

    PubMed Central

    Karaköse, Esra; Geiger, Tamar; Flynn, Kevin; Lorenz-Baath, Katrin; Zent, Roy; Mann, Matthias; Fässler, Reinhard

    2015-01-01

    ABSTRACT PINCH-1 is a LIM-only domain protein that forms a ternary complex with integrin-linked kinase (ILK) and parvin (to form the IPP complex) downstream of integrins. Here, we demonstrate that PINCH-1 (also known as Lims1) gene ablation in the epidermis of mice caused epidermal detachment from the basement membrane, epidermal hyperthickening and progressive hair loss. PINCH-1-deficient keratinocytes also displayed profound adhesion, spreading and migration defects in vitro that were substantially more severe than those of ILK-deficient keratinocytes indicating that PINCH-1 also exerts functions in an ILK-independent manner. By isolating the PINCH-1 interactome, the LIM-domain-containing and actin-binding protein epithelial protein lost in neoplasm (EPLIN, also known as LIMA1) was identified as a new PINCH-1-associated protein. EPLIN localized, in a PINCH-1-dependent manner, to integrin adhesion sites of keratinocytes in vivo and in vitro and its depletion severely attenuated keratinocyte spreading and migration on collagen and fibronectin without affecting PINCH-1 levels in focal adhesions. Given that the low PINCH-1 levels in ILK-deficient keratinocytes were sufficient to recruit EPLIN to integrin adhesions, our findings suggest that PINCH-1 regulates integrin-mediated adhesion of keratinocytes through the interactions with ILK as well as EPLIN. PMID:25609703

  3. An immunodominant membrane protein (Imp) of 'Candidatus Phytoplasma mali' binds to plant actin.

    PubMed

    Boonrod, K; Munteanu, B; Jarausch, B; Jarausch, W; Krczal, G

    2012-07-01

    The phytopathogenic, cell-wall-less phytoplasmas exhibit a dual life cycle: they multiply in the phloem of their host plant and in the body of their insect vector. Their membrane proteins are in direct contact with both hosts and are supposed to play a crucial role in the phytoplasma spread within the plant as well as by the insect vector. Three types of nonhomologous but highly abundant and immunodominant membrane proteins (IDP) have been identified within the phytoplasmas: Amp, IdpA, and Imp. Although recent results indicate that Amp is involved in vector specificity interacting with insect proteins such as actin, little is known about the interaction of IDP with the plant. We could demonstrate that transiently expressed Imp of 'Candidatus Phytoplasma mali' as well as the Imp without transmembrane domain (Imp▴Tm) bind with plant actins in vivo. Moreover, in vitro co-sediment and binding assays showed that Escherichia coli-expressed recombinant Imp▴Tm-His binds to both G- and F-actins isolated from rabbit muscle. Transgenic plants expressing Imp- or Imp▴Tm-green fluorescent protein did not exhibit any remarkable change of phenotype compared with the wild-type plant. These results indicate that Imp specifically binds to plant actin and a role of Imp-actin binding in phytoplasma motility is hypothesized. PMID:22432876

  4. Estrogen Regulates Protein Synthesis and Actin Polymerization in Hippocampal Neurons through Different Molecular Mechanisms

    PubMed Central

    Briz, Victor; Baudry, Michel

    2014-01-01

    Estrogen rapidly modulates hippocampal synaptic plasticity by activating selective membrane-associated receptors. Reorganization of the actin cytoskeleton and stimulation of mammalian target of rapamycin (mTOR)-mediated protein synthesis are two major events required for the consolidation of hippocampal long-term potentiation and memory. Estradiol regulates synaptic plasticity by interacting with both processes, but the underlying molecular mechanisms are not yet fully understood. Here, we used acute rat hippocampal slices to analyze the mechanisms underlying rapid changes in mTOR activity and actin polymerization elicited by estradiol. Estradiol-induced mTOR phosphorylation was preceded by rapid and transient activation of both extracellular signal-regulated kinase (ERK) and protein kinase B (Akt) and by phosphatase and tensin homolog (PTEN) degradation. These effects were prevented by calpain and ERK inhibitors. Estradiol-induced mTOR stimulation did not require activation of classical estrogen receptors (ER), as specific ERα and ERβ agonists (PPT and DPN, respectively) failed to mimic this effect, and ER antagonists could not block it. Estradiol rapidly activated both RhoA and p21-activated kinase (PAK). Furthermore, a specific inhibitor of RhoA kinase (ROCK), H1152, and a potent and specific PAK inhibitor, PF-3758309, blocked estradiol-induced cofilin phosphorylation and actin polymerization. ER antagonists also blocked these effects of estrogen. Consistently, both PPT and DPN stimulated PAK and cofilin phosphorylation as well as actin polymerization. Finally, the effects of estradiol on actin polymerization were insensitive to protein synthesis inhibitors, but its stimulation of mTOR activity was impaired by latrunculin A, a drug that disrupts actin filaments. Taken together, our results indicate that estradiol regulates local protein synthesis and cytoskeletal reorganization via different molecular mechanisms and signaling pathways. PMID:24611062

  5. Protein 4.1, a component of the erythrocyte membrane skeleton and its related homologue proteins forming the protein 4.1/FERM superfamily.

    PubMed

    Diakowski, Witold; Grzybek, Michał; Sikorski, Aleksander F

    2006-01-01

    The review is focused on the domain structure and function of protein 4.1, one of the proteins belonging to the membrane skeleton. The protein 4.1 of the red blood cells (4.1R) is a multifunctional protein that localizes to the membrane skeleton and stabilizes erythrocyte shape and membrane mechanical properties, such as deformability and stability, via lateral interactions with spectrin, actin, glycophorin C and protein p55. Protein 4.1 binding is modulated through the action of kinases and/or calmodulin-Ca2+. Non-erythroid cells express the 4.1R homologues: 4.1G (general type), 4.1B (brain type), and 4.1N (neuron type), and the whole group belongs to the protein 4.1 superfamily, which is characterized by the presence of a highly conserved FERM domain at the N-terminus of the molecule. Proteins 4.1R, 4.1G, 4.1N and 4.1B are encoded by different genes. Most of the 4.1 superfamily proteins also contain an actin-binding domain. To date, more than 40 members have been identified. They can be divided into five groups: protein 4.1 molecules, ERM proteins, talin-related molecules, protein tyrosine phosphatase (PTPH) proteins and NBL4 proteins. We have focused our attention on the main, well known representatives of 4.1 superfamily and tried to choose the proteins which are close to 4.1R or which have distinct functions. 4.1 family proteins are not just linkers between the plasma membrane and membrane skeleton; they also play an important role in various processes. Some, such as focal adhesion kinase (FAK), non-receptor tyrosine kinase that localizes to focal adhesions in adherent cells, play the role in cell adhesion. The other members control or take part in tumor suppression, regulation of cell cycle progression, inhibition of cell proliferation, downstream signaling of the glutamate receptors, and establishment of cell polarity; some are also involved in cell proliferation, cell motility, and/or cell-to-cell communication. PMID:17219717

  6. Biological role and structural mechanism of twinfilin–capping protein interaction

    PubMed Central

    Falck, Sandra; Paavilainen, Ville O; Wear, Martin A; Grossmann, J Günter; Cooper, John A; Lappalainen, Pekka

    2004-01-01

    Twinfilin and capping protein (CP) are highly conserved actin-binding proteins that regulate cytoskeletal dynamics in organisms from yeast to mammals. Twinfilin binds actin monomer, while CP binds the barbed end of the actin filament. Remarkably, twinfilin and CP also bind directly to each other, but the mechanism and role of this interaction in actin dynamics are not defined. Here, we found that the binding of twinfilin to CP does not affect the binding of either protein to actin. Furthermore, site-directed mutagenesis studies revealed that the CP-binding site resides in the conserved C-terminal tail region of twinfilin. The solution structure of the twinfilin–CP complex supports these conclusions. In vivo, twinfilin's binding to both CP and actin monomer was found to be necessary for twinfilin's role in actin assembly dynamics, based on genetic studies with mutants that have defined biochemical functions. Our results support a novel model for how sequential interactions between actin monomers, twinfilin, CP, and actin filaments promote cytoskeletal dynamics. PMID:15282541

  7. Drosophila Hook-Related Protein (Girdin) Is Essential for Sensory Dendrite Formation

    PubMed Central

    Ha, Andrew; Polyanovsky, Andrey; Avidor-Reiss, Tomer

    2015-01-01

    The dendrite of the sensory neuron is surrounded by support cells and is composed of two specialized compartments: the inner segment and the sensory cilium. How the sensory dendrite is formed and maintained is not well understood. Hook-related proteins (HkRP) like Girdin, DAPLE, and Gipie are actin-binding proteins, implicated in actin organization and in cell motility. Here, we show that the Drosophila melanogaster single member of the Hook-related protein family, Girdin, is essential for sensory dendrite formation and function. Mutations in girdin were identified during a screen for fly mutants with no mechanosensory function. Physiological, morphological, and ultrastructural studies of girdin mutant flies indicate that the mechanosensory neurons innervating external sensory organs (bristles) initially form a ciliated dendrite that degenerates shortly after, followed by the clustering of their cell bodies. Importantly, we observed that Girdin is expressed transiently during dendrite morphogenesis in three previously unidentified actin-based structures surrounding the inner segment tip and the sensory cilium. These actin structures are largely missing in girdin mutant. Defects in cilia are observed in other sensory organs such as those mediating olfaction and taste, suggesting that Girdin has a general role in forming sensory dendrites in Drosophila. These suggest that Girdin functions temporarily within the sensory organ and that this function is essential for the formation of the sensory dendrites via actin structures. PMID:26058848

  8. Divergent Evolution of CHD3 Proteins Resulted in MOM1 Refining Epigenetic Control in Vascular Plants

    PubMed Central

    Čaikovski, Marian; Yokthongwattana, Chotika; Habu, Yoshiki; Nishimura, Taisuke; Mathieu, Olivier; Paszkowski, Jerzy

    2008-01-01

    Arabidopsis MOM1 is required for the heritable maintenance of transcriptional gene silencing (TGS). Unlike many other silencing factors, depletion of MOM1 evokes transcription at selected loci without major changes in DNA methylation or histone modification. These loci retain unusual, bivalent chromatin properties, intermediate to both euchromatin and heterochromatin. The structure of MOM1 previously suggested an integral nuclear membrane protein with chromatin-remodeling and actin-binding activities. Unexpected results presented here challenge these presumed MOM1 activities and demonstrate that less than 13% of MOM1 sequence is necessary and sufficient for TGS maintenance. This active sequence encompasses a novel Conserved MOM1 Motif 2 (CMM2). The high conservation suggests that CMM2 has been the subject of strong evolutionary pressure. The replacement of Arabidopsis CMM2 by a poplar motif reveals its functional conservation. Interspecies comparison suggests that MOM1 proteins emerged at the origin of vascular plants through neo-functionalization of the ubiquitous eukaryotic CHD3 chromatin remodeling factors. Interestingly, despite the divergent evolution of CHD3 and MOM1, we observed functional cooperation in epigenetic control involving unrelated protein motifs and thus probably diverse mechanisms. PMID:18725928

  9. Molecular characterization of a troponin I-like protein from the hard tick Haemaphysalis longicornis.

    PubMed

    You, M; Xuan, X; Tsuji, N; Kamio, T; Igarashi, I; Nagasawa, H; Mikami, T; Fujisaki, K

    2001-12-01

    A cDNA expression library prepared from mRNA of Haemaphysalis longicornis (H. longicornis) was screened with a H. longicornis-infested rabbit serum. A cDNA encoding 27/30kDa proteins was cloned and designated P27/30 gene. The predicted amino acid sequence of the P27/30 gene shows a rather high homology (58% amino acid identities and 11% amino acid similarity) with Drosophila melanogaster troponin I clone E2. H. longicornis P27/30 possesses amino acid sequence of actin-binding domains of troponin I at the amino acid residues 128-148, suggesting that H. longicornis P27/30 is a troponin I-like protein. By immunoblot analysis, mouse anti-recombinant P27/30 serum reacted with major constituent protein bands in extracts of adult ticks, and also immunoreacted with muscle, cuticle, gut, and salivary gland in H. longicornis ticks. Moreover, immunohistochemistry using the anti-P27/30 serum showed a strong reactivity in muscle, suggesting that native P27/30 is expressed abundantly in that tissue. PMID:11719070

  10. Decabrominated diphenyl ether (BDE-209) and/or BDE-47 exposure alters protein expression in purified neural stem/progenitor cells determined by proteomics analysis.

    PubMed

    Song, Jie; Li, Zhi-hua; He, Yu-Tian; Liu, Chuan-Xin; Sun, Bin; Zhang, Chun-Fang; Zeng, Jie; Du, Pei-Li; Zhang, Hui-Li; Yu, Yan-hong; Chen, Dun-Jin

    2014-04-01

    Polybrominateddiphenyl ethers (PBDEs) are widely utilized as the additive brominated flame retardants in electronic devices, furniture, plastics, rubber foam, and textiles, which exhibit many negative biological effects, especially potential toxic effects on neurodevelopment. In the present study, we applied a proteomics approach to study the effects of decabromodiphenyl ether (BDE-209) and/or tetrabromodiphenyl ether (BDE-47) on the expression of proteins extracted from neural stem/progenitor cells and further explored mechanisms on neurodevelopmental toxicity. We sub-cultured 3-4 generations of neural stem/progenitor cells which were exposed to BDE-209 and/or BDE-47. After a 72-h exposure, we applied two-dimensional gel (2-DE) to identify differentially expressed proteins and matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS) to determine the protein identity of 25 spots. Western blot analysis was applied to determine the expression of cofilin-1 and vimentin. A total of 39 differential expression protein spots were identified by 2-DE after BDE-209 and/or BDE-47 exposure in the neural stem/progenitor cells, and 19 differentially expressed proteins were identified by MALDI-TOF-MS. Western blot analysis revealed that cofilin-1 and vimentin were differentially expressed in all groups. Expression of both proteins was decreased when the neural stem/progenitor cells were exposed to BDE-209 and were absent when exposed to both BDE-47 and BDE-209. BDE-209 and/or BDE-47 might alter the expression of some proteins of neural stem/progenitor cells. Nineteen proteins were identified by MALDI-TOF-MS, which will provide a useful basis for further study of the mechanisms underlying PBDE-mediated neurotoxicity. PMID:24239914

  11. Actin Interacting Protein1 and Actin Depolymerizing Factor Drive Rapid Actin Dynamics in Physcomitrella patens[W

    PubMed Central

    Augustine, Robert C.; Pattavina, Kelli A.; Tüzel, Erkan; Vidali, Luis; Bezanilla, Magdalena

    2011-01-01

    The remodeling of actin networks is required for a variety of cellular processes in eukaryotes. In plants, several actin binding proteins have been implicated in remodeling cortical actin filaments (F-actin). However, the extent to which these proteins support F-actin dynamics in planta has not been tested. Using reverse genetics, complementation analyses, and cell biological approaches, we assessed the in vivo function of two actin turnover proteins: actin interacting protein1 (AIP1) and actin depolymerizing factor (ADF). We report that AIP1 is a single-copy gene in the moss Physcomitrella patens. AIP1 knockout plants are viable but have reduced expansion of tip-growing cells. AIP1 is diffusely cytosolic and functions in a common genetic pathway with ADF to promote tip growth. Specifically, ADF can partially compensate for loss of AIP1, and AIP1 requires ADF for function. Consistent with a role in actin remodeling, AIP1 knockout lines accumulate F-actin bundles, have fewer dynamic ends, and have reduced severing frequency. Importantly, we demonstrate that AIP1 promotes and ADF is essential for cortical F-actin dynamics. PMID:22003077

  12. 12S-lipoxygenase protein associates with {alpha}-actin fibers in human umbilical artery vascular smooth muscle cells

    SciTech Connect

    Weisinger, Gary . E-mail: gary_w@tasmc.health.gov.il; Limor, Rona; Marcus-Perlman, Yonit; Knoll, Esther; Kohen, Fortune; Schinder, Vera; Firer, Michael; Stern, Naftali

    2007-05-11

    The current study sets out to characterize the intracellular localization of the platelet-type 12S-lipoxygenase (12-LO), an enzyme involved in angiotensin-II induced signaling in vascular smooth muscle cells (VSMC). Immunohistochemical analysis of VSMC in vitro or human umbilical arteries in vivo showed a clear cytoplasmic localization. On immunogold electron microscopy, 12-LO was found primarily associated with cytoplasmic VSMC muscle fibrils. Upon angiotensin-II treatment of cultured VSMC, immunoprecipitated 12-LO was found bound to {alpha}-actin, a component of the cytoplasmic myofilaments. 12-LO/{alpha}-actin binding was blocked by VSMC pretreatment with the 12-LO inhibitors, baicalien or esculetine and the protein synthesis inhibitor, cycloheximide. Moreover, the binding of 12-LO to {alpha}-actin was not associated with 12-LO serine or tyrosine phosphorylation. These observations suggest a previously unrecognized angiotensin-II dependent protein interaction in VSMC through which 12-LO protein may be trafficked, for yet undiscovered purposes towards the much more abundantly expressed cytoskeletal protein {alpha}-actin.

  13. Reaching Out to Send a Message: Proteins Associated with Neurite Outgrowth and Neurotransmission are Altered with Age in the Long-Lived Naked Mole-Rat.

    PubMed

    Triplett, Judy C; Swomley, Aaron M; Kirk, Jessime; Grimes, Kelly M; Lewis, Kaitilyn N; Orr, Miranda E; Rodriguez, Karl A; Cai, Jian; Klein, Jon B; Buffenstein, Rochelle; Butterfield, D Allan

    2016-07-01

    Aging is the greatest risk factor for developing neurodegenerative diseases, which are associated with diminished neurotransmission as well as neuronal structure and function. However, several traits seemingly evolved to avert or delay age-related deterioration in the brain of the longest-lived rodent, the naked mole-rat (NMR). The NMR remarkably also exhibits negligible senescence, maintaining an extended healthspan for ~75 % of its life span. Using a proteomic approach, statistically significant changes with age in expression and/or phosphorylation levels of proteins associated with neurite outgrowth and neurotransmission were identified in the brain of the NMR and include: cofilin-1; collapsin response mediator protein 2; actin depolymerizing factor; spectrin alpha chain; septin-7; syntaxin-binding protein 1; synapsin-2 isoform IIB; and dynamin 1. We hypothesize that such changes may contribute to the extended lifespan and healthspan of the NMR. PMID:26935741

  14. Accommodation of structural rearrangements in the huntingtin-interacting protein 1 coiled-coil domain

    SciTech Connect

    Wilbur, Jeremy D.; Hwang, Peter K.; Brodsky, Frances M.; Fletterick, Robert J.

    2010-03-01

    Variable packing interaction related to the conformational flexibility within the huntingtin-interacting protein 1 coiled coil domain. Huntingtin-interacting protein 1 (HIP1) is an important link between the actin cytoskeleton and clathrin-mediated endocytosis machinery. HIP1 has also been implicated in the pathogenesis of Huntington’s disease. The binding of HIP1 to actin is regulated through an interaction with clathrin light chain. Clathrin light chain binds to a flexible coiled-coil domain in HIP1 and induces a compact state that is refractory to actin binding. To understand the mechanism of this conformational regulation, a high-resolution crystal structure of a stable fragment from the HIP1 coiled-coil domain was determined. The flexibility of the HIP1 coiled-coil region was evident from its variation from a previously determined structure of a similar region. A hydrogen-bond network and changes in coiled-coil monomer interaction suggest that the HIP1 coiled-coil domain is uniquely suited to allow conformational flexibility.

  15. Reduced Myelin Basic Protein and Actin-Related Gene Expression in Visual Cortex in Schizophrenia

    PubMed Central

    Matthews, Paul R.; Eastwood, Sharon L.; Harrison, Paul J.

    2012-01-01

    Most brain gene expression studies of schizophrenia have been conducted in the frontal cortex or hippocampus. The extent to which alterations occur in other cortical regions is not well established. We investigated primary visual cortex (Brodmann area 17) from the Stanley Neuropathology Consortium collection of tissue from 60 subjects with schizophrenia, bipolar disorder, major depression, or controls. We first carried out a preliminary array screen of pooled RNA, and then used RT-PCR to quantify five mRNAs which the array identified as differentially expressed in schizophrenia (myelin basic protein [MBP], myelin-oligodendrocyte glycoprotein [MOG], β-actin [ACTB], thymosin β-10 [TB10], and superior cervical ganglion-10 [SCG10]). Reduced mRNA levels were confirmed by RT-PCR for MBP, ACTB and TB10. The MBP reduction was limited to transcripts containing exon 2. ACTB and TB10 mRNAs were also decreased in bipolar disorder. None of the transcripts were altered in subjects with major depression. Reduced MBP mRNA in schizophrenia replicates findings in other brain regions and is consistent with oligodendrocyte involvement in the disorder. The decreases in expression of ACTB, and the actin-binding protein gene TB10, suggest changes in cytoskeletal organisation. The findings confirm that the primary visual cortex shows molecular alterations in schizophrenia and extend the evidence for a widespread, rather than focal, cortical pathophysiology. PMID:22675524

  16. Actin Cytoskeleton Manipulation by Effector Proteins Secreted by Diarrheagenic Escherichia coli Pathotypes

    PubMed Central

    Navarro-Garcia, Fernando; Serapio-Palacios, Antonio; Ugalde-Silva, Paul; Tapia-Pastrana, Gabriela; Chavez-Dueñas, Lucia

    2013-01-01

    The actin cytoskeleton is a dynamic structure necessary for cell and tissue organization, including the maintenance of epithelial barriers. Disruption of the epithelial barrier coincides with alterations of the actin cytoskeleton in several disease states. These disruptions primarily affect the paracellular space, which is normally regulated by tight junctions. Thereby, the actin cytoskeleton is a common and recurring target of bacterial virulence factors. In order to manipulate the actin cytoskeleton, bacteria secrete and inject toxins and effectors to hijack the host cell machinery, which interferes with host-cell pathways and with a number of actin binding proteins. An interesting model to study actin manipulation by bacterial effectors is Escherichia coli since due to its genome plasticity it has acquired diverse genetic mobile elements, which allow having different E. coli varieties in one bacterial species. These E. coli pathotypes, including intracellular and extracellular bacteria, interact with epithelial cells, and their interactions depend on a specific combination of virulence factors. In this paper we focus on E. coli effectors that mimic host cell proteins to manipulate the actin cytoskeleton. The study of bacterial effector-cytoskeleton interaction will contribute not only to the comprehension of the molecular causes of infectious diseases but also to increase our knowledge of cell biology. PMID:23509714

  17. Adenylyl cyclase-associated protein 1 in metastasis of squamous cell carcinoma of the head and neck and non-small cell lung cancer

    NASA Astrophysics Data System (ADS)

    Kakurina, G. V.; Kolegova, E. S.; Cheremisina, O. V.; Zavyalov, A. A.; Shishkin, D. A.; Kondakova, I. V.; Choinzonov, E. L.

    2016-08-01

    Progression of tumors and metastasis in particular is one of the main reasons of the high mortality rate among cancer patients. The primary role in developing metastases plays cell locomotion which requires remodeling of the actin cytoskeleton. Form, dynamics, localization and mechanical properties of the actin cytoskeleton are regulated by a variety of actin-binding proteins, which include the adenylyl cyclase-associated protein 1 (CAP1). The study is devoted to the investigation of CAP1 level depending on the presence or absence of metastases in patients with squamous cell carcinoma of the head and neck (SCCHN) and non-small cell lung cancer (NSCLC). The results show the contribution of CAP1 to SCCHN and NSCLC progression. We detected the connection between the tissue protein CAP1 level and the stage of NSCLC and SCCHN disease. Also the levels of the CAP1 protein in tissues of primary tumors and metastases in lung cancer were different. Our data showed that CAP is important in the development of metastases, which suggests further perspectives in the study of this protein for projecting metastasis of NSCLC and SCCHN.

  18. Arabidopsis Microtubule-Destabilizing Protein 25 Functions in Pollen Tube Growth by Severing Actin Filaments[W

    PubMed Central

    Qin, Tao; Liu, Xiaomin; Li, Jiejie; Sun, Jingbo; Song, Leina; Mao, Tonglin

    2014-01-01

    The formation of distinct actin filament arrays in the subapical region of pollen tubes is crucial for pollen tube growth. However, the molecular mechanisms underlying the organization and dynamics of the actin filaments in this region remain to be determined. This study shows that Arabidopsis thaliana MICROTUBULE-DESTABILIZING PROTEIN25 (MDP25) has the actin filament–severing activity of an actin binding protein. This protein negatively regulated pollen tube growth by modulating the organization and dynamics of actin filaments in the subapical region of pollen tubes. MDP25 loss of function resulted in enhanced pollen tube elongation and inefficient fertilization. MDP25 bound directly to actin filaments and severed individual actin filaments, in a manner that was dramatically enhanced by Ca2+, in vitro. Analysis of a mutant that bears a point mutation at the Ca2+ binding sites demonstrated that the subcellular localization of MDP25 was determined by cytosolic Ca2+ level in the subapical region of pollen tubes, where MDP25 was disassociated from the plasma membrane and moved into the cytosol. Time-lapse analysis showed that the F-actin-severing frequency significantly decreased and a high density of actin filaments was observed in the subapical region of mdp25-1 pollen tubes. This study reveals a mechanism whereby calcium enhances the actin filament–severing activity of MDP25 in the subapical region of pollen tubes to modulate pollen tube growth. PMID:24424096

  19. Human CASK/LIN-2 Binds Syndecan-2 and Protein 4.1 and Localizes to the Basolateral Membrane of Epithelial Cells

    PubMed Central

    Cohen, Alexandra R.; Wood, Daniel F.; Marfatia, Shirin M.; Walther, Zenta; Chishti, Athar H.; Anderson, James Melvin

    1998-01-01

    In Caenorhabditis elegans, mutations in the lin-2 gene inactivate the LET-23 receptor tyrosine kinase/Ras/MAP kinase pathway required for vulval cell differentiation. One function of LIN-2 is to localize LET-23 to the basal membrane domain of vulval precursor cells. LIN-2 belongs to the membrane-associated guanylate kinase family of proteins. We have cloned and characterized the human homolog of LIN-2, termed hCASK, and Northern and Western blot analyses reveal that it is ubiquitously expressed. Indirect immunofluorescence localizes CASK to distinct lateral and/or basal plasma membrane domains in different epithelial cell types. We detect in a yeast two-hybrid screen that the PDZ domain of hCASK binds to the heparan sulfate proteoglycan syndecan-2. This interaction is confirmed using in vitro binding assays and immunofluorescent colocalization. Furthermore, we demonstrate that hCASK binds the actin-binding protein 4.1. Syndecans are known to bind extracellular matrix, and to form coreceptor complexes with receptor tyrosine kinases. We speculate that CASK mediates a link between the extracellular matrix and the actin cytoskeleton via its interaction with syndecan and with protein 4.1. Like other membrane-associated guanylate kinases, its multidomain structure enables it to act as a scaffold at the membrane, potentially recruiting multiple proteins and coordinating signal transduction. PMID:9660868

  20. The Disruption of the Cytoskeleton during Semaphorin 3A induced Growth Cone Collapse Correlates with Differences in Actin Organization and Associated Binding Proteins

    PubMed Central

    Brown, Jacquelyn A; Bridgman, Paul C

    2010-01-01

    Repulsive guidance cues induce growth cone collapse or collapse and retraction. Collapse results from disruption and loss of the actin cytoskeleton. Actin rich regions of growth cones contain binding proteins that influence filament organization, such as Arp2/3, cortactin, and fascin, but little is known about the role that these proteins play in collapse. Here we show that Semaphorin 3A (Sema 3A), which is repulsive to mouse dorsal root ganglion neurons, has unequal effects on actin binding proteins and their associated filaments. The immunofluorescence staining intensity of Arp-2 and cortactin decreases relative to total protein, while in unextracted growth cones fascin increases. Fascin and myosin IIB staining redistribute and show increased overlap. The degree of actin filament loss during collapse correlates with filament superstructures detected by rotary shadow electron microscopy. Collapse results in the loss of branched f-actin meshworks, while actin bundles are partially retained to varying degrees. Taken together with the known affects of Sema 3A on actin, this suggests a model for collapse that follows a sequence; depolymerization of actin meshworks followed by partial depolymerization of fascin associated actin bundles and their movement to the neurite to complete collapse. The relocated fascin associated actin bundles may provide the substrate for actomyosin contractions that produce retraction. PMID:19513995

  1. Safety pharmacology, toxicology and pharmacokinetic assessment of human Gc globulin (vitamin D binding protein).

    PubMed

    Pihl, Tina Holberg; Jørgensen, Charlotte Svaerke; Santoni-Rugiu, Eric; Leifsson, Páll Skúli; Hansen, Erik Wind; Laursen, Inga; Houen, Gunnar

    2010-11-01

    Gc globulin is an important protein of the plasma actin-scavenger system. As such, it has been shown to bind free actin and prevent hypercoagulation and shock in patients with massive actin release resulting from severe tissue injuries. Treatment of such patients with Gc globulin could therefore potentially be life-saving. This article presents pre-clinical toxicology experiments conducted on purified plasma-derived human Gc globulin. The Gc globulin formulation was shown to be stable for at least 4 years with full retention of actin-binding capacity. In vitro studies did not reveal activation of the kallikrein system or the complement system and cellular studies showed no toxic effects on a variety of human cell lines. In vivo studies showed no acute toxic effects in mice, rats or guinea pigs upon intravenous infusion. A 14-day local tolerance study in rabbits showed no adverse effects, and 14-day toxicity studies in rats and horses did not show any unwanted reactions. In a 14-day toxicology study in beagle dogs, formation of antibodies was seen and in the end of the study period, three out of four dogs showed clinical immunological reactions, which could be ascribed to the formation of antibodies. The half-life, T, for human Gc globulin was 12 hr in rats, 16 hr in horses and 30 hr in dogs. The safety profile of plasma-derived Gc globulin is concluded to be consistent to that required for use in man. PMID:20560927

  2. Ezrin/radixin/moesin proteins differentially regulate endothelial hyperpermeability after thrombin.

    PubMed

    Adyshev, Djanybek M; Dudek, Steven M; Moldobaeva, Nurgul; Kim, Kyung-mi; Ma, Shwu-Fan; Kasa, Anita; Garcia, Joe G N; Verin, Alexander D

    2013-08-01

    Endothelial cell (EC) barrier disruption induced by inflammatory agonists such as thrombin leads to potentially lethal physiological dysfunction such as alveolar flooding, hypoxemia, and pulmonary edema. Thrombin stimulates paracellular gap and F-actin stress fiber formation, triggers actomyosin contraction, and alters EC permeability through multiple mechanisms that include protein kinase C (PKC) activation. We previously have shown that the ezrin, radixin, and moesin (ERM) actin-binding proteins differentially participate in sphingosine-1 phosphate-induced EC barrier enhancement. Phosphorylation of a conserved threonine residue in the COOH-terminus of ERM proteins causes conformational changes in ERM to unmask binding sites and is considered a hallmark of ERM activation. In the present study we test the hypothesis that ERM proteins are phosphorylated on this critical threonine residue by thrombin-induced signaling events and explore the role of the ERM family in modulating thrombin-induced cytoskeletal rearrangement and EC barrier function. Thrombin promotes ERM phosphorylation at this threonine residue (ezrin Thr567, radixin Thr564, moesin Thr558) in a PKC-dependent fashion and induces translocation of phosphorylated ERM to the EC periphery. Thrombin-induced ERM threonine phosphorylation is likely synergistically mediated by protease-activated receptors PAR1 and PAR2. Using the siRNA approach, depletion of either moesin alone or of all three ERM proteins significantly attenuates thrombin-induced increase in EC barrier permeability (transendothelial electrical resistance), cytoskeletal rearrangements, paracellular gap formation, and accumulation of phospho-myosin light chain. In contrast, radixin depletion exerts opposing effects on these indexes. These data suggest that ERM proteins play important differential roles in the thrombin-induced modulation of EC permeability, with moesin promoting barrier dysfunction and radixin opposing it. PMID:23729486

  3. Isolation of a strawberry gene fragment encoding an actin depolymerizing factor-like protein from genotypes resistant to Colletotrichum acutatum.

    PubMed

    Ontivero, Marta; Zamora, Gustavo Martínez; Salazar, Sergio; Ricci, Juan Carlos Díaz; Castagnaro, Atilio Pedro

    2011-12-01

    Actin depolymerizing factors (ADFs) have been recently implicated in plant defense against pathogenic fungi, associated with the cytoskeletal rearrangements that contribute to establish an effective barrier against fungal ingress. In this work, we identified a DNA fragment corresponding to a part of a gene predicted to encode an ADF-like protein in genotypes of Fragaria ananassa resistant to the fungus Colletotrichum acutatum. Bulked segregant analysis combined with AFLP was used to identify polymorphisms linked to resistance in hybrids derived from the cross between the resistant cultivar 'Sweet Charlie' and the susceptible cultivar 'Pájaro'. The sequence of one out of three polymorphic bands detected showed significant BLASTX hits to ADF proteins from other plants. Two possible exons were identified and bioinformatic analysis revealed the presence of the ADF homology domain with two actin-binding sites, an N-terminal phosphorylation site, and a nuclear localization signal. In addition to its possible application in strawberry breeding programs, these finding may contribute to investigate the role of ADFs in plant resistance against fungi. PMID:22107362

  4. Pathophysiology of protein aggregation and extended phenotyping in filaminopathy

    PubMed Central

    Serdaroglu-Oflazer, Piraye; Leber, Yvonne; Odgerel, Zagaa; van der Ven, Peter F. M.; Olivé, Montse; Ferrer, Isidro; Onipe, Adekunle; Mihaylov, Mariya; Bilbao, Juan M.; Lee, Hee S.; Höhfeld, Jörg; Djinović-Carugo, Kristina; Kong, Kester; Tegenthoff, Martin; Peters, Sören A.; Stenzel, Werner; Vorgerd, Matthias; Goldfarb, Lev G.; Fürst, Dieter O.

    2012-01-01

    Mutations in FLNC cause two distinct types of myopathy. Disease associated with mutations in filamin C rod domain leading to expression of a toxic protein presents with progressive proximal muscle weakness and shows focal destructive lesions of polymorphous aggregates containing desmin, myotilin and other proteins in the affected myofibres; these features correspond to the profile of myofibrillar myopathy. The second variant associated with mutations in the actin-binding domain of filamin C is characterized by weakness of distal muscles and morphologically by non-specific myopathic features. A frameshift mutation in the filamin C rod domain causing haploinsufficiency was also found responsible for distal myopathy with some myofibrillar changes but no protein aggregation typical of myofibrillar myopathies. Controversial data accumulating in the literature require re-evaluation and comparative analysis of phenotypes associated with the position of the FLNC mutation and investigation of the underlying disease mechanisms. This is relevant and necessary for the refinement of diagnostic criteria and developing therapeutic approaches. We identified a p.W2710X mutation in families originating from ethnically diverse populations and re-evaluated a family with a p.V930_T933del mutation. Analysis of the expanded database allows us to refine clinical and myopathological characteristics of myofibrillar myopathy caused by mutations in the rod domain of filamin C. Biophysical and biochemical studies indicate that certain pathogenic mutations in FLNC cause protein misfolding, which triggers aggregation of the mutant filamin C protein and subsequently involves several other proteins. Immunofluorescence analyses using markers for the ubiquitin–proteasome system and autophagy reveal that the affected muscle fibres react to protein aggregate formation with a highly increased expression of chaperones and proteins involved in proteasomal protein degradation and autophagy. However

  5. Pathophysiology of protein aggregation and extended phenotyping in filaminopathy.

    PubMed

    Kley, Rudolf A; Serdaroglu-Oflazer, Piraye; Leber, Yvonne; Odgerel, Zagaa; van der Ven, Peter F M; Olivé, Montse; Ferrer, Isidro; Onipe, Adekunle; Mihaylov, Mariya; Bilbao, Juan M; Lee, Hee S; Höhfeld, Jörg; Djinović-Carugo, Kristina; Kong, Kester; Tegenthoff, Martin; Peters, Sören A; Stenzel, Werner; Vorgerd, Matthias; Goldfarb, Lev G; Fürst, Dieter O

    2012-09-01

    Mutations in FLNC cause two distinct types of myopathy. Disease associated with mutations in filamin C rod domain leading to expression of a toxic protein presents with progressive proximal muscle weakness and shows focal destructive lesions of polymorphous aggregates containing desmin, myotilin and other proteins in the affected myofibres; these features correspond to the profile of myofibrillar myopathy. The second variant associated with mutations in the actin-binding domain of filamin C is characterized by weakness of distal muscles and morphologically by non-specific myopathic features. A frameshift mutation in the filamin C rod domain causing haploinsufficiency was also found responsible for distal myopathy with some myofibrillar changes but no protein aggregation typical of myofibrillar myopathies. Controversial data accumulating in the literature require re-evaluation and comparative analysis of phenotypes associated with the position of the FLNC mutation and investigation of the underlying disease mechanisms. This is relevant and necessary for the refinement of diagnostic criteria and developing therapeutic approaches. We identified a p.W2710X mutation in families originating from ethnically diverse populations and re-evaluated a family with a p.V930_T933del mutation. Analysis of the expanded database allows us to refine clinical and myopathological characteristics of myofibrillar myopathy caused by mutations in the rod domain of filamin C. Biophysical and biochemical studies indicate that certain pathogenic mutations in FLNC cause protein misfolding, which triggers aggregation of the mutant filamin C protein and subsequently involves several other proteins. Immunofluorescence analyses using markers for the ubiquitin-proteasome system and autophagy reveal that the affected muscle fibres react to protein aggregate formation with a highly increased expression of chaperones and proteins involved in proteasomal protein degradation and autophagy. However, there

  6. Basement membrane protein ladinin-1 and the MIF-CD44-β1 integrin signaling axis are implicated in laryngeal cancer metastasis.

    PubMed

    Klobučar, Marko; Sedić, Mirela; Gehrig, Peter; Grossmann, Jonas; Bilić, Mario; Kovač-Bilić, Lana; Pavelić, Krešimir; Kraljević Pavelić, Sandra

    2016-10-01

    Laryngeal squamous cell carcinoma (LSCC) is the most common form of malignant disease in the head and neck region characterized by frequent occurrence of metastases in the neck lymph nodes early in the disease onset. In the presented study, we performed quantitative proteomic profiling of patient-matched primary tumor and adjacent non-tumorous tissues derived from metastatic LSCC as to identify new protein candidates with potential diagnostic and therapeutic significance. Obtained results revealed for the first time involvement of the basement membrane protein ladinin-1 in laryngeal cancer metastases. Alterations in the cellular microenvironment that propel metastatic events in laryngeal cancer include activation of MIF-CD44-β1 integrin signal transduction pathway and induction of downstream signaling mediated by NF-κB and Src tyrosine kinase, which ultimately impinge on cytoskeletal dynamics and architecture resulting in increased cellular motility and invasiveness. In this context, particularly interesting finding is upregulation of several actin-binding proteins novel to laryngeal cancer pathogenesis including coronin-1C and plastin-2, whose functional significance in laryngeal carcinogenesis has yet to be established. We also detected for the first time a complete loss of afamin in metastatic laryngeal cancer tissues, which warrants further studies into its use as a possible marker for monitoring disease progression and/or treatment outcome. PMID:27460703

  7. Novel recognition motifs and biological functions of the RNA-binding protein HuD revealed by genome-wide identification of its targets

    PubMed Central

    Bolognani, Federico; Contente-Cuomo, Tania; Perrone-Bizzozero, Nora I.

    2010-01-01

    HuD is a neuronal ELAV-like RNA-binding protein (RBP) involved in nervous system development, regeneration, and learning and memory. This protein stabilizes mRNAs by binding to AU-rich instability elements (AREs) in their 3′ unstranslated regions (3′ UTR). To isolate its in vivo targets, messenger ribonucleoprotein (mRNP) complexes containing HuD were first immunoprecipitated from brain extracts and directly bound mRNAs identified by subsequent GST-HuD pull downs and microarray assays. Using the 3′ UTR sequences of the most enriched targets and the known sequence restrictions of the HuD ARE-binding site, we discovered three novel recognition motifs. Motifs 2 and 3 are U-rich whereas motif 1 is C-rich. In vitro binding assays indicated that HuD binds motif 3 with the highest affinity, followed by motifs 2 and 1, with less affinity. These motifs were found to be over-represented in brain mRNAs that are upregulated in HuD overexpressor mice, supporting the biological function of these sequences. Gene ontology analyses revealed that HuD targets are enriched in signaling pathways involved in neuronal differentiation and that many of these mRNAs encode other RBPs, translation factors and actin-binding proteins. These findings provide further insights into the post-transcriptional mechanisms by which HuD promotes neural development and synaptic plasticity. PMID:19846595

  8. Organization of the human protein 4.1 genomic locus: New insights into the tissue-specific alternative splicing of the pre-mRNA

    SciTech Connect

    Baklouti, F. ||; Huang, Shu-Ching; Benz, E.J. Jr. |

    1997-02-01

    Protein 4.1 is a globular 80-kDa component of the erythrocyte membrane skeleton that enhances spectrin-actin interaction via its internal 10-kDa domain. Previous studies have shown that protein 4.1 mRNA is expressed as multiple alternatively spliced isoforms, resulting from the inclusion or exclusion of small cassette sequences called motifs. By tissue screening for protein 4.1 isoforms, we have observed new features of an already complex pattern of alternative splicing within the spectrin/actin binding domain. In particular, we found a new 51-nt exon that is present almost exclusively in muscle tissue. In addition, we have isolated multiple genomic clones spanning over 200 kb, containing the entire erythroid and nonerythroid coding sequence of the human locus. The exon/intron structure has now been characterized; with the exception of a 17-nt motif, all of the alternatively spliced motifs correspond to individual exons. The 3{prime}-untranslated region (UTR) has also been completely sequenced using various PCR and genomic-sequencing methods. The 3{prime} UTR, over 3 kb, accounts for one-half of the mature mRNA. 83 refs., 8 figs., 1 tab.

  9. Novel β-Propeller of the BTB-Kelch Protein Krp1 Provides a Binding Site for Lasp-1 That Is Necessary for Pseudopodial Extension*♦

    PubMed Central

    Gray, Christopher H.; McGarry, Lynn C.; Spence, Heather J.; Riboldi-Tunnicliffe, Alan; Ozanne, Bradford W.

    2009-01-01

    Kelch-related protein 1 (Krp1) is up-regulated in oncogene-transformed fibroblasts. The Kelch repeats interact directly with the actin-binding protein Lasp-1 in membrane ruffles at the tips of pseudopodia, where both proteins are necessary for pseudopodial elongation. Herein, we investigate the molecular basis for this interaction. Probing an array of overlapping decapeptides of Rattus norvegicus (Rat) Krp1 with recombinant Lasp-1 revealed two binding sites; one (317YDPMENECYLT327) precedes the first of five Kelch repeats, and the other (563TEVNDIWKYEDD574) is in the last of the five Kelch repeats. Mutational analysis established that both binding sites are necessary for Krp1-Lasp-1 interaction in vitro and function in vivo. The crystal structure of the C-terminal domain of rat Krp1 (amino acids 289–606) reveals that both binding sites are brought into close proximity by the formation of a novel six-bladed β-propeller, where the first blade is not formed by a Kelch repeat. PMID:19726686

  10. TAGLN2 regulates T cell activation by stabilizing the actin cytoskeleton at the immunological synapse

    PubMed Central

    Na, Bo-Ra; Kim, Hye-Ran; Piragyte, Indre; Oh, Hyun-Mee; Kwon, Min-Sung; Akber, Uroos; Lee, Hyun-Su; Park, Do-Sim; Song, Woo Keun; Park, Zee-Yong; Im, Sin-Hyeog; Rho, Mun-Chual; Hyun, Young-Min; Kim, Minsoo

    2015-01-01

    The formation of an immunological synapse (IS) requires tight regulation of actin dynamics by many actin polymerizing/depolymerizing proteins. However, the significance of actin stabilization at the IS remains largely unknown. In this paper, we identify a novel function of TAGLN2—an actin-binding protein predominantly expressed in T cells—in stabilizing cortical F-actin, thereby maintaining F-actin contents at the IS and acquiring LFA-1 (leukocyte function-associated antigen-1) activation after T cell receptor stimulation. TAGLN2 blocks actin depolymerization and competes with cofilin both in vitro and in vivo. Knockout of TAGLN2 (TAGLN2−/−) reduced F-actin content and destabilized F-actin ring formation, resulting in decreased cell adhesion and spreading. TAGLN2−/− T cells displayed weakened cytokine production and cytotoxic effector function. These findings reveal a novel function of TAGLN2 in enhancing T cell responses by controlling actin stability at the IS. PMID:25869671

  11. Loss of cortactin causes endothelial barrier dysfunction via disturbed adrenomedullin secretion and actomyosin contractility

    PubMed Central

    García Ponce, Alexander; Citalán Madrid, Alí F.; Vargas Robles, Hilda; Chánez Paredes, Sandra; Nava, Porfirio; Betanzos, Abigail; Zarbock, Alexander; Rottner, Klemens; Vestweber, Dietmar; Schnoor, Michael

    2016-01-01

    Changes in vascular permeability occur during inflammation and the actin cytoskeleton plays a crucial role in regulating endothelial cell contacts and permeability. We demonstrated recently that the actin-binding protein cortactin regulates vascular permeability via Rap1. However, it is unknown if the actin cytoskeleton contributes to increased vascular permeability without cortactin. As we consistently observed more actin fibres in cortactin-depleted endothelial cells, we hypothesised that cortactin depletion results in increased stress fibre contractility and endothelial barrier destabilisation. Analysing the contractile machinery, we found increased ROCK1 protein levels in cortactin-depleted endothelium. Concomitantly, myosin light chain phosphorylation was increased while cofilin, mDia and ERM were unaffected. Secretion of the barrier-stabilising hormone adrenomedullin, which activates Rap1 and counteracts actomyosin contractility, was reduced in plasma from cortactin-deficient mice and in supernatants of cortactin-depleted endothelium. Importantly, adrenomedullin administration and ROCK1 inhibition reduced actomyosin contractility and rescued the effect on permeability provoked by cortactin deficiency in vitro and in vivo. Our data suggest a new role for cortactin in controlling actomyosin contractility with consequences for endothelial barrier integrity. PMID:27357373

  12. Loss of cortactin causes endothelial barrier dysfunction via disturbed adrenomedullin secretion and actomyosin contractility.

    PubMed

    García Ponce, Alexander; Citalán Madrid, Alí F; Vargas Robles, Hilda; Chánez Paredes, Sandra; Nava, Porfirio; Betanzos, Abigail; Zarbock, Alexander; Rottner, Klemens; Vestweber, Dietmar; Schnoor, Michael

    2016-01-01

    Changes in vascular permeability occur during inflammation and the actin cytoskeleton plays a crucial role in regulating endothelial cell contacts and permeability. We demonstrated recently that the actin-binding protein cortactin regulates vascular permeability via Rap1. However, it is unknown if the actin cytoskeleton contributes to increased vascular permeability without cortactin. As we consistently observed more actin fibres in cortactin-depleted endothelial cells, we hypothesised that cortactin depletion results in increased stress fibre contractility and endothelial barrier destabilisation. Analysing the contractile machinery, we found increased ROCK1 protein levels in cortactin-depleted endothelium. Concomitantly, myosin light chain phosphorylation was increased while cofilin, mDia and ERM were unaffected. Secretion of the barrier-stabilising hormone adrenomedullin, which activates Rap1 and counteracts actomyosin contractility, was reduced in plasma from cortactin-deficient mice and in supernatants of cortactin-depleted endothelium. Importantly, adrenomedullin administration and ROCK1 inhibition reduced actomyosin contractility and rescued the effect on permeability provoked by cortactin deficiency in vitro and in vivo. Our data suggest a new role for cortactin in controlling actomyosin contractility with consequences for endothelial barrier integrity. PMID:27357373

  13. Functional manipulation of a calcium-binding protein from Entamoeba histolytica guided by paramagnetic NMR.

    PubMed

    Rout, Ashok K; Patel, Sunita; Somlata; Shukla, Manish; Saraswathi, Deepa; Bhattacharya, Alok; Chary, Kandala V R

    2013-08-01

    EhCaBP1, one of the calcium-binding proteins from Entamoeba histolytica, is a two-domain EF-hand protein. The two domains of EhCaBP1 are structurally and functionally different from each other. However, both domains are required for structural stability and a full range of functional diversity. Analysis of sequence and structure of EhCaBP1 and other CaBPs indicates that the C-terminal domain of EhCaBP1 possesses a unique structure compared with other family members. This had been attributed to the absence of a Phe-Phe interaction between highly conserved Phe residues at the -4 position in EF-hand III (F[-4]; Tyr(81)) and at the 13th position in EF-hand IV (F[+13]; Phe(129)) of the C-terminal domain. Against this backdrop, we mutated the Tyr residue at the -4th position of EF III to the Phe residue (Y81F), to bring in the Phe-Phe interaction and understand the nature of structural and functional changes in the protein by NMR spectroscopy, molecular dynamics (MD) simulation, isothermal titration calorimetry (ITC), and biological assays, such as imaging and actin binding. The Y81F mutation in EhCaBP1 resulted in a more compact structure for the C-terminal domain of the mutant as in the case of calmodulin and troponin C. The compact structure is favored by the presence of a π-π interaction between Phe(81) and Phe(129) along with several hydrophobic interactions of Phe(81), which are not seen in the wild-type protein. Furthermore, the biological assays reveal preferential membrane localization of the mutant, loss of its colocalization with actin in the phagocytic cups, whereas retaining its ability to bind G- and F-actin. PMID:23782698

  14. Cytoskeletal binding proteins distinguish cultured dental follicle cells and periodontal ligament cells.

    PubMed

    Li, Jie; Li, Hui; Tian, Ye; Yang, Yaling; Chen, Guoqing; Guo, Weihua; Tian, Weidong

    2016-07-01

    Human dental follicle cells (DFCs) and periodontal ligament cells (PDLCs) derived from the ectomesenchymal tissue, have been shown to exhibit stem/progenitor cell properties and the ability to induce tissue regeneration. Stem cells in dental follicle differentiate into cementoblasts, periodontal ligament fibroblasts and osteoblasts, these cells form cementum, periodontal ligament and alveolar bone, respectively. While stem cells in dental follicle are a precursor to periodontal ligament fibroblasts, the molecular changes that distinguish cultured DFCs from PDLCs are still unknown. In this study, we have compared the immunophenotypic features and cell cycle status of the two cell lines. The results suggest that DFCs and PDLCs displayed similar features related to immunophenotype and cell cycle. Then we employed an isobaric tag for relative and absolute quantitation (iTRAQ) proteomics strategy to reveal the molecular differences between the two cell types. A total of 2138 proteins were identified and 39 of these proteins were consistently differentially expressed between DFCs and PDLCs. Gene ontology analyses revealed that the protein subsets expressed higher in PDLCs were related to actin binding, cytoskeletal protein binding, and structural constituent of muscle. Upon validation by real-time PCR, western blotting, and immunofluorescence staining. Tropomyosin 1 (TPM1) and caldesmon 1 (CALD1) were expressed higher in PDLCs than in DFCs. Our results suggested that PDLCs display enhanced actin cytoskeletal dynamics relative to DFCs while DFCs may exhibit a more robust antioxidant defense ability relative to PDLCs. This study expands our knowledge of the cultured DFCs and PDLCs proteome and provides new insights into possible mechanisms responsible for the different biological features observed in each cell type. PMID:26708290

  15. Arabidopsis VILLIN5, an Actin Filament Bundling and Severing Protein, Is Necessary for Normal Pollen Tube Growth[W

    PubMed Central

    Zhang, Hua; Qu, Xiaolu; Bao, Chanchan; Khurana, Parul; Wang, Qiannan; Xie, Yurong; Zheng, Yiyan; Chen, Naizhi; Blanchoin, Laurent; Staiger, Christopher J.; Huang, Shanjin

    2010-01-01

    A dynamic actin cytoskeleton is essential for pollen germination and tube growth. However, the molecular mechanisms underlying the organization and turnover of the actin cytoskeleton in pollen remain poorly understood. Villin plays a key role in the formation of higher-order structures from actin filaments and in the regulation of actin dynamics in eukaryotic cells. It belongs to the villin/gelsolin/fragmin superfamily of actin binding proteins and is composed of six gelsolin-homology domains at its core and a villin headpiece domain at its C terminus. Recently, several villin family members from plants have been shown to sever, cap, and bundle actin filaments in vitro. Here, we characterized a villin isovariant, Arabidopsis thaliana VILLIN5 (VLN5), that is highly and preferentially expressed in pollen. VLN5 loss-of-function retarded pollen tube growth and sensitized actin filaments in pollen grains and tubes to latrunculin B. In vitro biochemical analyses revealed that VLN5 is a typical member of the villin family and retains a full suite of activities, including barbed-end capping, filament bundling, and calcium-dependent severing. The severing activity was confirmed with time-lapse evanescent wave microscopy of individual actin filaments in vitro. We propose that VLN5 is a major regulator of actin filament stability and turnover that functions in concert with oscillatory calcium gradients in pollen and therefore plays an integral role in pollen germination and tube growth. PMID:20807879

  16. RBFOX2 Promotes Protein 4.1R Exon 16 Selection via U1 snRNP Recruitment

    PubMed Central

    Ou, Alexander C.; Park, Jennie; Yu, Faye; Yu, Brian; Lee, Angela; Yang, Guang; Zhou, Anyu; Benz, Edward J.

    2012-01-01

    The erythroid differentiation-specific splicing switch of protein 4.1R exon 16, which encodes a spectrin/actin-binding peptide critical for erythrocyte membrane stability, is modulated by the differentiation-induced splicing factor RBFOX2. We have now characterized the mechanism by which RBFOX2 regulates exon 16 splicing through the downstream intronic element UGCAUG. Exon 16 possesses a weak 5′ splice site (GAG/GTTTGT), which when strengthened to a consensus sequence (GAG/GTAAGT) leads to near-total exon 16 inclusion. Impaired RBFOX2 binding reduces exon 16 inclusion in the context of the native weak 5′ splice site, but not the engineered strong 5′ splice site, implying that RBFOX2 achieves its effect by promoting utilization of the weak 5′ splice site. We further demonstrate that RBFOX2 increases U1 snRNP recruitment to the weak 5′ splice site through direct interaction between its C-terminal domain (CTD) and the zinc finger region of U1C and that the CTD is required for the effect of RBFOX2 on exon 16 splicing. Our data suggest a novel mechanism for exon 16 5′ splice site activation in which the binding of RBFOX2 to downstream intronic splicing enhancers stabilizes the pre-mRNA–U1 snRNP complex through interactions with U1C. PMID:22083953

  17. Systematic mutational analysis of the amino-terminal domain of the Listeria monocytogenes ActA protein reveals novel functions in actin-based motility.

    PubMed

    Lauer, P; Theriot, J A; Skoble, J; Welch, M D; Portnoy, D A

    2001-12-01

    The Listeria monocytogenes ActA protein acts as a scaffold to assemble and activate host cell actin cytoskeletal factors at the bacterial surface, resulting in directional actin polymerization and propulsion of the bacterium through the cytoplasm. We have constructed 20 clustered charged-to-alanine mutations in the NH2-terminal domain of ActA and replaced the endogenous actA gene with these molecular variants. These 20 clones were evaluated in several biological assays for phenotypes associated with particular amino acid changes. Additionally, each protein variant was purified and tested for stimulation of the Arp2/3 complex, and a subset was tested for actin monomer binding. These specific mutations refined the two regions involved in Arp2/3 activation and suggest that the actin-binding sequence of ActA spans 40 amino acids. We also identified a 'motility rate and cloud-to-tail transition' region in which nine contiguous mutations spanning amino acids 165-260 caused motility rate defects and changed the ratio of intracellular bacteria associated with actin clouds and comet tails without affecting Arp2/3 activation. Several unusual motility phenotypes were associated with amino acid changes in this region, including altered paths through the cytoplasm, discontinuous actin tails in host cells and the tendency to 'skid' or dramatically change direction while moving. These unusual phenotypes illustrate the complexity of ActA functions that control the actin-based motility of L. monocytogenes. PMID:11886549

  18. Anillin Regulates Neuronal Migration and Neurite Growth by Linking RhoG to the Actin Cytoskeleton.

    PubMed

    Tian, Dong; Diao, Min; Jiang, Yuxiang; Sun, Lingfei; Zhang, Yan; Chen, Zhucheng; Huang, Shanjin; Ou, Guangshuo

    2015-05-01

    Neuronal migration and neurite growth are essential events in neural development, but it remains unclear how guidance cues are transduced through receptors to the actin cytoskeleton, which powers these processes. We report that a cytokinetic scaffold protein, Anillin, is redistributed to the leading edge of the C. elegans Q neuroblast during cell migration and neurite growth. To bypass the requirement for Anillin in cytokinesis, we used the somatic CRISPR-Cas9 technique to generate conditional mutations in Anillin. We demonstrate that Anillin regulates cell migration and growth cone extension by stabilizing the F-actin network at the leading edge. Our biochemical analysis shows that the actin-binding domain of Anillin is sufficient to stabilize F-actin by antagonizing the F-actin severing activity of Cofilin. We further uncover that the active form of RhoG/MIG-2 directly binds to Anillin and recruits it to the leading edge. Our results reveal a novel pathway in which Anillin transduces the RhoG signal to the actin cytoskeleton during neuronal migration and neurite growth. PMID:25843030

  19. A novel neuron-enriched homolog of the erythrocyte membrane cytoskeletal protein 4.1.

    PubMed

    Walensky, L D; Blackshaw, S; Liao, D; Watkins, C C; Weier, H U; Parra, M; Huganir, R L; Conboy, J G; Mohandas, N; Snyder, S H

    1999-08-01

    We report the molecular cloning and characterization of 4.1N, a novel neuronal homolog of the erythrocyte membrane cytoskeletal protein 4.1 (4.1R). The 879 amino acid protein shares 70, 36, and 46% identity with 4.1R in the defined membrane-binding, spectrin-actin-binding, and C-terminal domains, respectively. 4.1N is expressed in almost all central and peripheral neurons of the body and is detected in embryonic neurons at the earliest stage of postmitotic differentiation. Like 4.1R, 4.1N has multiple splice forms as evidenced by PCR and Western analysis. Whereas the predominant 4.1N isoform identified in brain is approximately 135 kDa, a smaller 100 kDa isoform is enriched in peripheral tissues. Immunohistochemical studies using a polyclonal 4.1N antibody revealed several patterns of neuronal staining, with localizations in the neuronal cell body, dendrites, and axons. In certain neuronal locations, including the granule cell layers of the cerebellum and dentate gyrus, a distinct punctate-staining pattern was observed consistent with a synaptic localization. In primary hippocampal cultures, mouse 4.1N is enriched at the discrete sites of synaptic contact, colocalizing with the postsynaptic density protein of 95 kDa (a postsynaptic marker) and glutamate receptor type 1 (an excitatory postsynaptic marker). By analogy with the roles of 4.1R in red blood cells, 4.1N may function to confer stability and plasticity to the neuronal membrane via interactions with multiple binding partners, including the spectrin-actin-based cytoskeleton, integral membrane channels and receptors, and membrane-associated guanylate kinases. PMID:10414974

  20. Allopregnanolone increases mature excitatory synapses along dendrites via protein kinase A signaling.

    PubMed

    Shimizu, H; Ishizuka, Y; Yamazaki, H; Shirao, T

    2015-10-01

    Allopregnanolone (APα; 5α-pregnan-3α-ol-20-one) is synthesized in both the periphery and central nervous system and is known to be a potent positive allosteric modulator of the GABAA receptor. Because APα was suggested to improve the symptoms of depression and Alzheimer's disease (AD), which involve synaptic dysfunction and loss, we examined whether APα affects excitatory synapses. Drebrin, which is an actin-binding protein, forms a unique stable actin structure in dendritic spines, and drebrin levels correlate positively with cognitive levels in AD and mild cognitive impairment. We investigated whether APα increases excitatory synapse density along dendrites of mature hippocampal neurons using drebrin-imaging-based evaluation of mature synapses. We prepared primary cultures of hippocampal neurons and either transfected them with GFP or immunostained them against drebrin. Morphological analysis of GFP-transfected neurons revealed that a 24-h exposure to 0.3 or 1 μM APα significantly increased dendritic spine density without any morphological changes to spines. Drebrin cluster density was also increased by 0.3 and 1 μM APα. The protein kinase A (PKA) inhibitor H-89 inhibited the APα-induced increase in drebrin cluster density. These data demonstrate that APα increases mature excitatory synapses via activation of PKA. Therefore, the PKA-cAMP response element-binding protein (CREB) signaling pathway is likely to be involved in the APα-induced increase of mature excitatory synapses. Another possibility is that the PKA-dependent increase in AMPA receptors at dendritic spines mediates the APα function. In conclusion, our study indicates that APα may improve neuropsychiatric disorder outcomes via increasing the numbers of mature excitatory synapses. PMID:26241343

  1. Proteomic analysis of pregnancy-related proteins from pig uterus endometrium during pregnancy

    PubMed Central

    2011-01-01

    Many important molecular events associated with implantation and development occur within the female reproductive tract, especially within the uterus endometrium, during pregnancy periods. The endometrium includes the mucosal lining of the uterus, which provides a suitable site for implantation and development of a fertilized egg and fetus. To date, the molecular cascades in the uterus endometrium during pregnancy periods in pigs have not been elucidated fully. In this study, we compared the functional regulated proteins in the endometrium during pregnancy periods with those in non-pregnant conditions and investigated changes in expression patterns during pregnancy (days 40, 70, and 93) using two-dimensional gel electrophoresis (2-DE) and western blotting. The functional regulated proteins were identified and discovered from differentially expressed proteins in the uterus endometrium during pregnancy. We discovered 820 protein spots in a proteomic analysis of uterus endometrium tissues with 2-DE gels. We identified 63 of the 98 proteins regulated differentially among non-pregnant and pregnant tissues (matched and unmatched spots). Interestingly, 10 of these 63 proteins are development-, cytoskeleton- and chaperon-related proteins such as transferrin, protein DJ-1, transgelin, galectin-1, septin 2, stathmin 1, cofilin 1, fascin 1, heat shock protein (HSP) 90β and HSP 27. The specific expression patterns of these proteins in the endometrium during pregnancy were confirmed by western blotting. Our results suggest that the expressions of these genes involved in endometrium function and endometrium development from early to late gestation are associated with the regulation of endometrium development for maintaining pregnancy. PMID:21791079

  2. Coronin 1B Regulates S1P-Induced Human Lung Endothelial Cell Chemotaxis: Role of PLD2, Protein Kinase C and Rac1 Signal Transduction

    PubMed Central

    Mohan, Vijay; Pendyala, Srikanth; He, Donghong; Ebenezer, David L.; Harijith, Anantha; Fu, Panfeng; Huang, Long Shuang; Bear, James E.; Garcia, Joe G. N.; Natarajan, Viswanathan

    2013-01-01

    Coronins are a highly conserved family of actin binding proteins that regulate actin-dependent processes such as cell motility and endocytosis. We found that treatment of human pulmonary artery endothelial cells (HPAECs) with the bioactive lipid, sphingosine-1-phosphate (S1P) rapidly stimulates coronin 1B translocation to lamellipodia at the cell leading edge, which is required for S1P-induced chemotaxis. Further, S1P-induced chemotaxis of HPAECs was attenuated by pretreatment with small interfering RNA (siRNA) targeting coronin 1B (∼36%), PLD2 (∼45%) or Rac1 (∼50%) compared to scrambled siRNA controls. Down regulation PLD2 expression by siRNA also attenuated S1P-induced coronin 1B translocation to the leading edge of the cell periphery while PLD1 silencing had no effect. Also, S1P-induced coronin 1B redistribution to cell periphery and chemotaxis was attenuated by inhibition of Rac1 and over-expression of dominant negative PKC δ, ε and ζ isoforms in HPAECs. These results demonstrate that S1P activation of PLD2, PKC and Rac1 is part of the signaling cascade that regulates coronin 1B translocation to the cell periphery and the ensuing cell chemotaxis. PMID:23667561

  3. 2E4 (Kaptin): A novel actin-associated protein from human blood platelets found in lamellipodia and the tips of the stereocilia of the inner ear

    PubMed Central

    Bearer, Elaine L.; Abraham, Manoj T.

    2010-01-01

    Actin – 2E4/kaptin – platelet activation – stereocilia – sensory epithelium Platelet activation, crucial for hemostasis, requires actin polymerization, yet the molecular mechanisms by which localized actin polymerization is mediated are not clear. Here we report the characterization of a novel actin-binding protein. 2E4, originally isolated from human blood platelets and likely to be involved in the actin rearrangements occurring during activation. 2E4 binds to filamentous (F)-actin by F-actin affinity chromatography and is eluted from F-actin affinity columns and extracted from cells with ATP. Its presence at the leading edge of platelets spread on glass and in the lamellipodia of motile fibroblasts suggests a role in actin dynamics. Using localization to obtain clues about function, we stained the sensory epithelium of the embryonic inner car to determine whether 2E4 is at the barbed end of actin filaments during their elongation. Indeed, 2E4 was present at the tips of the elongating stereocilium. 2E4 is novel by DNA sequence and has no identifiable structural motifs. Its unusual amino acid sequence, its ATP-sensitive actin association and its location at sites of actin polymerization in cells suggest 2E4 plays a unique role in the actin rearrangements that accompany platelet activation and stereocilia formation. PMID:10099934

  4. Filamin A-interacting protein (FILIP) is a region-specific modulator of myosin 2b and controls spine morphology and NMDA receptor accumulation

    PubMed Central

    Yagi, Hideshi; Nagano, Takashi; Xie, Min-Jue; Ikeda, Hiroshi; Kuroda, Kazuki; Komada, Munekazu; Iguchi, Tokuichi; Tariqur, Rahman M.; Morikubo, Soichi; Noguchi, Koichi; Murase, Kazuyuki; Okabe, Masaru; Sato, Makoto

    2014-01-01

    Learning and memory depend on morphological and functional changes to neural spines. Non-muscle myosin 2b regulates actin dynamics downstream of long-term potentiation induction. However, the mechanism by which myosin 2b is regulated in the spine has not been fully elucidated. Here, we show that filamin A-interacting protein (FILIP) is involved in the control of neural spine morphology and is limitedly expressed in the brain. FILIP bound near the ATPase domain of non-muscle myosin heavy chain IIb, an essential component of myosin 2b, and modified the function of myosin 2b by interfering with its actin-binding activity. In addition, FILIP altered the subcellular distribution of myosin 2b in spines. Moreover, subunits of the NMDA receptor were differently distributed in FILIP-expressing neurons, and excitation propagation was altered in FILIP-knockout mice. These results indicate that FILIP is a novel, region-specific modulator of myosin 2b. PMID:25220605

  5. Filamin A-interacting protein (FILIP) is a region-specific modulator of myosin 2b and controls spine morphology and NMDA receptor accumulation.

    PubMed

    Yagi, Hideshi; Nagano, Takashi; Xie, Min-Jue; Ikeda, Hiroshi; Kuroda, Kazuki; Komada, Munekazu; Iguchi, Tokuichi; Tariqur, Rahman M; Morikubo, Soichi; Noguchi, Koichi; Murase, Kazuyuki; Okabe, Masaru; Sato, Makoto

    2014-01-01

    Learning and memory depend on morphological and functional changes to neural spines. Non-muscle myosin 2b regulates actin dynamics downstream of long-term potentiation induction. However, the mechanism by which myosin 2b is regulated in the spine has not been fully elucidated. Here, we show that filamin A-interacting protein (FILIP) is involved in the control of neural spine morphology and is limitedly expressed in the brain. FILIP bound near the ATPase domain of non-muscle myosin heavy chain IIb, an essential component of myosin 2b, and modified the function of myosin 2b by interfering with its actin-binding activity. In addition, FILIP altered the subcellular distribution of myosin 2b in spines. Moreover, subunits of the NMDA receptor were differently distributed in FILIP-expressing neurons, and excitation propagation was altered in FILIP-knockout mice. These results indicate that FILIP is a novel, region-specific modulator of myosin 2b. PMID:25220605

  6. Overexpression of gelsolin-like actin-capping protein is associated with progression of lung adenocarcinoma.

    PubMed

    Shao, Fangchun; Zhang, Ruifeng; Don, Liangliang; Ying, Kejing

    2011-01-01

    Gelsolin-like actin-capping protein (CapG), a ubiquitous actin-binding protein, has been shown to play a critical role in regulating the migration ability of cells. In this study, we investigated CapG expression in lung cancer cell lines under hypoxia and evaluated the effect of CapG on the migration ability of these cells. We also analyzed the expression of CapG in a total of 75 patients with lung adenocarcinoma by immunohistochemistry. Our results showed that hypoxia increased the expression of CapG in the human lung cancer cell lines, A549 and H358. Knockdown of CapG expression with small interfering RNA led to a decrease in the migration ability of these cell lines. These results indicate that CapG expression is upregulated in lung cancer cell lines under hypoxia and that CapG may contribute to the migration ability of lung cancer cells. Moreover, the excised lung adenocarcinoma tissues showed significantly increased immunoreactivity for CapG, compared to the adjacent tumor-free tissues. Importantly, overexpression of CapG is significantly associated with male sex (χ(2) = 5.195, p = 0.033) and lymph node metastasis (χ(2) = 5.58, p = 0.021). Likewise, CapG overexpression was observed with advanced tumor stages (III and IV, 16/31), compared with early tumor stages (I and II, 14/44), but the difference was not statistically significant. These results suggest that overexpression of CapG may be associated with progression of lung adenocarcinoma. In conclusion, CapG may be a promising target for therapy and a potential biomarker for predicting the prognosis of lung adenocarcinoma. PMID:21908955

  7. Oxytocin Increases Neurite Length and Expression of Cytoskeletal Proteins Associated with Neuronal Growth.

    PubMed

    Lestanova, Z; Bacova, Z; Kiss, A; Havranek, T; Strbak, V; Bakos, J

    2016-06-01

    Neuropeptide oxytocin acts as a growth and differentiation factor; however, its effects on neurite growth are poorly understood. The aims of the present study were (1) to evaluate time effects of oxytocin on expression of nestin and MAP2; (2) to measure the effect of oxytocin on gene expression of β-actin, vimentin, cofilin, and drebrin; and (3) to measure changes in neurite length and number in response to oxytocin/oxytocin receptor antagonist L-371,257. Exposure of SH-SY5Y cells to 1 μM oxytocin resulted in a significant increase in gene expression and protein levels of nestin after 12, 24, and 48 h. Oxytocin treatment induced no changes in gene expression of MAP2; however, a decrease of protein levels was observed in all time intervals. Gene expression of β-actin, vimentin, and drebrin increased in response to oxytocin. Oxytocin induced significant elongation of neurites after 12, 24, and 48 h. No change in neurite length was observed in the presence of the combination of retinoic acid and oxytocin receptor antagonist L-371,257. Oxytocin treatment for 12 h increased the number of neurites. Overall, the present data suggest that oxytocin contributes to the regulation of expression of cytoskeletal proteins associated with growth of neuronal cones and induces neurite elongation mediated by oxytocin receptors at least in certain types of neuronal cells. PMID:26474566

  8. Arginine kinase: differentiation of gene expression and protein activity in the red imported fire ant, Solenopsis invicta.

    PubMed

    Wang, Haichuan; Zhang, Lan; Zhang, Lee; Lin, Qin; Liu, Nannan

    2009-02-01

    Arginine kinase (AK), a primary enzyme in cell metabolism and adenosine 5'-triphosphate (ATP)-consuming processes, plays an important role in cellular energy metabolism and maintaining constant ATP levels in invertebrate cells. In order to identify genes that are differentially expressed between larvae and adults, queens and workers, and female alates (winged) and queens (wingless), AK cDNA was obtained from the red imported fire ant. The cDNA sequence of the gene has open reading frames of 1065 nucleotides, encoding a protein of 355 amino acid residues that includes the substrate recognition region, the signature sequence pattern of ATP:guanidino kinases, and an "actinin-type" actin binding domain. Northern blot analysis and protein activity analysis demonstrated that the expression of the AK gene and its protein activity were developmentally, caste specifically, and tissue specifically regulated in red imported fire ants with a descending order of worker> alate (winged adult) female> alate (winged adult) male> larvae> worker pupae approximately alate pupae. These results suggest a different demand for energy-consumption and production in the different castes of the red imported fire ant, which may be linked to their different missions and physiological activities in the colonies. The highest level of the AK gene expression and activity was identified in head tissue of both female alates and workers and thorax tissue of workers, followed by thorax tissue of female alates and abdomen tissue of male alates, suggesting the main tissues or cells in these body parts, such as brain, neurons and muscles, which have been identified as the major tissues and/or cells that display high and variable rates of energy turnover in other organisms, play a key role in energy production and its utilization in the fire ant. In contrast, in the male alate, the highest AK expression and activity were found in the abdomen, suggesting that here energy demand may relate to sperm formation

  9. Cardiac muscle cell cytoskeletal protein 4.1: analysis of transcripts and subcellular location--relevance to membrane integrity, microstructure, and possible role in heart failure.

    PubMed

    Taylor-Harris, Pamela M; Keating, Lisa A; Maggs, Alison M; Phillips, Gareth W; Birks, Emma J; Franklin, Rodney C G; Yacoub, Magdi H; Baines, Anthony J; Pinder, Jennifer C

    2005-03-01

    The spectrin-based cytoskeleton assembly has emerged as a major player in heart functioning; however, cardiac protein 4.1, a key constituent, is uncharacterized. Protein 4.1 evolved to protect cell membranes against mechanical stresses and to organize membrane microstructure. 4.1 Proteins are multifunctional and, among other activities, link integral/signaling proteins on the plasma and internal membranes with the spectrin-based cytoskeleton. Four genes, EPB41, EPB41L1, EPB41L2, and EPB41L3 encode proteins 4.1R, 4.1N, 4.1G, and 4.1B, respectively. All are extensively spliced. Different isoforms are expressed according to tissue and developmental state, individual function being controlled through inclusion/exclusion of interactive domains. We have defined mouse and human cardiac 4.1 transcripts; other than 4. 1B in humans, all genes show activity. Cardiac transcripts constitutively include conserved FERM and C-terminal domains; both interact with membrane-bound signaling/transport/cell adhesion molecules. Variable splicing within and adjacent to the central spectrin/actin-binding domain enables regulation of cytoskeleton-binding activity. A novel heart-specific exon occurs in human 4.1G, but not in mouse. Immunofluorescence reveals 4.1 staining within mouse cardiomyocytes; thus, both at the plasma membrane and, interdigitated with sarcomeric myosin, across myofibrils in regions close to the sarcoplasmic reticulum. These are all regions to which spectrin locates. 4.1R in human heart shows similar distribution; however, there is limited plasma membrane staining. We conclude that cardiac 4.1s are highly regulated in their ability to crosslink plasma/integral cell membranes with the spectrin-actin cytoskeleton. We speculate that over the repetitive cycles of heart muscle contraction and relaxation, 4.1s are likely to locate, support, and coordinate functioning of key membrane-bound macromolecular assemblies. PMID:15834631

  10. A role for complexes of survival of motor neurons (SMN) protein with gemins and profilin in neurite-like cytoplasmic extensions of cultured nerve cells

    SciTech Connect

    Sharma, Aarti; Lambrechts, Anja; Le thi Hao; Le, Thanh T.; Sewry, Caroline A.; Ampe, Christophe; Burghes, Arthur H.M.; Morris, Glenn E. . E-mail: glenn.morris@rjah.nhs.uk

    2005-09-10

    Spinal muscular atrophy (SMA) is caused by reduced levels of SMN (survival of motor neurons protein) and consequent loss of motor neurons. SMN is involved in snRNP transport and nuclear RNA splicing, but axonal transport of SMN has also been shown to occur in motor neurons. SMN also binds to the small actin-binding protein, profilin. We now show that SMN and profilin II co-localise in the cytoplasm of differentiating rat PC12 cells and in neurite-like extensions, especially at their growth cones. Many components of known SMN complexes were also found in these extensions, including gemin2 (SIP-1), gemin6, gemin7 and unrip (unr-interacting protein). Coilin p80 and Sm core protein immunoreactivity, however, were seen only in the nucleus. SMN is known to associate with {beta}-actin mRNA and specific hnRNPs in axons and in neurite extensions of cultured nerve cells, and SMN also stimulates neurite outgrowth in cultures. Our results are therefore consistent with SMN complexes, rather than SMN alone, being involved in the transport of actin mRNPs along the axon as in the transport of snRNPs into the nucleus by similar SMN complexes. Antisense knockdown of profilin I and II isoforms inhibited neurite outgrowth of PC12 cells and caused accumulation of SMN and its associated proteins in cytoplasmic aggregates. BIAcore studies demonstrated a high affinity interaction of SMN with profilin IIa, the isoform present in developing neurons. Pathogenic missense mutations in SMN, or deletion of exons 5 and 7, prevented this interaction. The interaction is functional in that SMN can modulate actin polymerisation in vitro by reducing the inhibitory effect of profilin IIa. This suggests that reduced SMN in SMA might cause axonal pathfinding defects by disturbing the normal regulation of microfilament growth by profilins.

  11. Role of Peroxiredoxin 1 and Peroxiredoxin 4 in Protection of Respiratory Syncytial Virus-Induced Cysteinyl Oxidation of Nuclear Cytoskeletal Proteins

    PubMed Central

    Jamaluddin, Mohammad; Wiktorowicz, John E.; Soman, Kizhake V.; Boldogh, Istvan; Forbus, Jeffrey D.; Spratt, Heidi; Garofalo, Roberto P.; Brasier, Allan R.

    2010-01-01

    The respiratory epithelium plays a central role in innate immunity by secreting networks of inflammatory mediators in response to respiratory syncytial virus (RSV) infection. Previous proteomic studies focusing on the host cellular response to RSV indicated the existence of a nuclear heat shock response and cytoplasmic depletion of antioxidant proteins in model type II-like airway epithelial cells. Here, we increased the depth of nuclear proteomic interrogation by using fluorescence difference labeling followed by liquid isoelectric focusing prefractionation/two-dimensional gel electrophoresis (2-DE) to identify an additional 41 proteins affected by RSV infection. Surprisingly, we found inducible oligomers and shifts in isoelectric points for peroxiredoxin 1 (Prdx-1), Prdx-3, and Prdx-4 isoforms without changes in their total abundance, indicating that Prdxs were being oxidized in response to RSV. To address the role of Prdx-1 and Prdx-4 in RSV infection, isoforms were selectively knocked down by small interfering RNA (siRNA) transfection. Cells lacking Prdx-1, Prdx-4, or both showed increased levels of reactive oxygen species formation and a higher level of protein carbonylation in response to RSV infection. Using a novel saturation fluorescence labeling 2-DE analysis, we showed that 15 unique proteins had enhanced oxidative modifications of at least >1.2-fold in the Prdx knockdowns in response to RSV, including annexin A2 and desmoplakin. Our results suggest that Prdx-1 and Prdx-4 are essential for preventing RSV-induced oxidative damage in a subset of nuclear intermediate filament and actin binding proteins in epithelial cells. PMID:20610706

  12. Role of peroxiredoxin 1 and peroxiredoxin 4 in protection of respiratory syncytial virus-induced cysteinyl oxidation of nuclear cytoskeletal proteins.

    PubMed

    Jamaluddin, Mohammad; Wiktorowicz, John E; Soman, Kizhake V; Boldogh, Istvan; Forbus, Jeffrey D; Spratt, Heidi; Garofalo, Roberto P; Brasier, Allan R

    2010-09-01

    The respiratory epithelium plays a central role in innate immunity by secreting networks of inflammatory mediators in response to respiratory syncytial virus (RSV) infection. Previous proteomic studies focusing on the host cellular response to RSV indicated the existence of a nuclear heat shock response and cytoplasmic depletion of antioxidant proteins in model type II-like airway epithelial cells. Here, we increased the depth of nuclear proteomic interrogation by using fluorescence difference labeling followed by liquid isoelectric focusing prefractionation/two-dimensional gel electrophoresis (2-DE) to identify an additional 41 proteins affected by RSV infection. Surprisingly, we found inducible oligomers and shifts in isoelectric points for peroxiredoxin 1 (Prdx-1), Prdx-3, and Prdx-4 isoforms without changes in their total abundance, indicating that Prdxs were being oxidized in response to RSV. To address the role of Prdx-1 and Prdx-4 in RSV infection, isoforms were selectively knocked down by small interfering RNA (siRNA) transfection. Cells lacking Prdx-1, Prdx-4, or both showed increased levels of reactive oxygen species formation and a higher level of protein carbonylation in response to RSV infection. Using a novel saturation fluorescence labeling 2-DE analysis, we showed that 15 unique proteins had enhanced oxidative modifications of at least >1.2-fold in the Prdx knockdowns in response to RSV, including annexin A2 and desmoplakin. Our results suggest that Prdx-1 and Prdx-4 are essential for preventing RSV-induced oxidative damage in a subset of nuclear intermediate filament and actin binding proteins in epithelial cells. PMID:20610706

  13. Avoiding artefacts when counting polymerized actin in live cells with LifeAct fused to fluorescent proteins.

    PubMed

    Courtemanche, Naomi; Pollard, Thomas D; Chen, Qian

    2016-06-01

    When tagged with a fluorescent protein, actin is not fully functional, so the LifeAct peptide fused to a fluorescent protein is widely used to localize actin filaments in live cells. However, we find that these fusion proteins have many concentration-dependent effects on actin assembly in vitro and in fission yeast cells. mEGFP-LifeAct inhibits actin assembly during endocytosis as well as assembly and constriction of the cytokinetic contractile ring. Purified mEGFP-LifeAct and LifeAct-mCherry bind actin filaments with Kd values of ∼10 μM. LifeAct-mCherry can promote actin filament nucleation and either promote or inhibit filament elongation. Both separately and together, profilin and formins suppress these effects. LifeAct-mCherry can also promote or inhibit actin filament severing by cofilin. These concentration-dependent effects mean that caution is necessary when overexpressing LifeAct fusion proteins to label actin filaments in cells. Therefore, we used low micromolar concentrations of tagged LifeAct to follow assembly and disassembly of actin filaments in cells. Careful titrations also gave an estimate of a peak of ∼190,000 actin molecules (∼500 μm) in the fission yeast contractile ring. These filaments shorten from ∼500 to ∼100 subunits as the ring constricts. PMID:27159499

  14. Modeling of protein binary complexes using structural mass spectrometry data

    PubMed Central

    Kamal, J.K. Amisha; Chance, Mark R.

    2008-01-01

    In this article, we describe a general approach to modeling the structure of binary protein complexes using structural mass spectrometry data combined with molecular docking. In the first step, hydroxyl radical mediated oxidative protein footprinting is used to identify residues that experience conformational reorganization due to binding or participate in the binding interface. In the second step, a three-dimensional atomic structure of the complex is derived by computational modeling. Homology modeling approaches are used to define the structures of the individual proteins if footprinting detects significant conformational reorganization as a function of complex formation. A three-dimensional model of the complex is constructed from these binary partners using the ClusPro program, which is composed of docking, energy filtering, and clustering steps. Footprinting data are used to incorporate constraints—positive and/or negative—in the docking step and are also used to decide the type of energy filter—electrostatics or desolvation—in the successive energy-filtering step. By using this approach, we examine the structure of a number of binary complexes of monomeric actin and compare the results to crystallographic data. Based on docking alone, a number of competing models with widely varying structures are observed, one of which is likely to agree with crystallographic data. When the docking steps are guided by footprinting data, accurate models emerge as top scoring. We demonstrate this method with the actin/gelsolin segment-1 complex. We also provide a structural model for the actin/cofilin complex using this approach which does not have a crystal or NMR structure. PMID:18042684

  15. Mutations in NEBL encoding the cardiac Z-disk protein nebulette are associated with various cardiomyopathies

    PubMed Central

    Tomasov, Pavol; Villard, Eric; Faludi, Reka; Melacini, Paola; Lossie, Janine; Lohmann, Nadine; Richard, Pascale; De Bortoli, Marzia; Angelini, Annalisa; Varga-Szemes, Akos; Sperling, Silke R.; Simor, Tamás; Veselka, Josef; Özcelik, Cemil; Charron, Philippe

    2016-01-01

    Introduction Transgenic mice overexpressing mutated NEBL, encoding the cardiac-specific Z-disk protein nebulette, develop severe cardiac phenotypes. Since cardiomyopathies are commonly familial and because mutations in a single gene may result in variable phenotypes, we tested the hypothesis that NEBL mutations are associated with cardiomyopathy. Material and methods We analyzed 389 patients, including cohorts of patients with dilated cardiomyopathy (DCM), hypertrophic cardiomyopathy (HCM), and left ventricular non-compaction cardiomyopathy (LVNC). The 28 coding exons of the NEBL gene were sequenced. Further bioinformatic analysis was used to distinguish variants. Results In total, we identified six very rare heterozygous missense mutations in NEBL in 7 different patients (frequency 1.8%) in highly conserved codons. The mutations were not detectable in 320 Caucasian sex-matched unrelated individuals without cardiomyopathy and 192 Caucasian sex-matched blood donors without heart disease. Known cardiomyopathy genes were excluded in these patients. The mutations p.H171R and p.I652L were found in 2 HCM patients. Further, p.Q581R and p.S747L were detected in 2 DCM patients, while the mutation p.A175T was identified independently in two unrelated patients with DCM. One LVNC patient carried the mutation p.P916L. All HCM and DCM related mutations were located in the nebulin-like repeats, domains responsible for actin binding. Interestingly, the mutation associated with LVNC was located in the C-terminal serine-rich linker region. Conclusions Our data suggest that NEBL mutations may cause various cardiomyopathies. We herein describe the first NEBL mutations in HCM and LVNC. Our findings underline the notion that the cardiomyopathies are true allelic diseases. PMID:27186169

  16. Association with PAK2 Enables Functional Interactions of Lentiviral Nef Proteins with the Exocyst Complex

    PubMed Central

    Imle, Andrea; Abraham, Libin; Tsopoulidis, Nikolaos; Hoflack, Bernard; Saksela, Kalle

    2015-01-01

    ABSTRACT Human immunodeficiency virus type 1 (HIV-1) Nef enhances virus replication and contributes to immune evasion in vivo, but the underlying molecular mechanisms remain incompletely defined. Nef interferes with host cell actin dynamics to restrict T lymphocyte responses to chemokine stimulation and T cell receptor engagement. This relies on the assembly of a labile multiprotein complex including the host kinase PAK2 that Nef usurps to phosphorylate and inactivate the actin-severing factor cofilin. Components of the exocyst complex (EXOC), an octameric protein complex involved in vesicular transport and actin remodeling, were recently reported to interact with Nef via the same molecular surface that mediates PAK2 association. Exploring the functional relevance of EXOC in Nef-PAK2 complex assembly/function, we found Nef-EXOC interactions to be specifically mediated by the PAK2 interface of Nef, to occur in infected human T lymphocytes, and to be conserved among lentiviral Nef proteins. In turn, EXOC was dispensable for direct downstream effector functions of Nef-associated PAK2. Surprisingly, PAK2 was essential for Nef-EXOC association, which required a functional Rac1/Cdc42 binding site but not the catalytic activity of PAK2. EXOC was dispensable for Nef functions in vesicular transport but critical for inhibition of actin remodeling and proximal signaling upon T cell receptor engagement. Thus, Nef exploits PAK2 in a stepwise mechanism in which its kinase activity cooperates with an adaptor function for EXOC to inhibit host cell actin dynamics. PMID:26350970

  17. Activation of the Low Molecular Weight Protein Tyrosine Phosphatase in Keratinocytes Exposed to Hyperosmotic Stress

    PubMed Central

    Cavalheiro, Renan P.; Machado, Daisy; Cruz, Bread L. G.; Paredes-Gamero, Edgar J.; Gomes-Marcondes, Maria C. C.; Zambuzzi, Willian F.; Vasques, Luciana; Nader, Helena B.; Souza, Ana Carolina S.; Justo, Giselle Z.

    2015-01-01

    Herein, we provide new contribution to the mechanisms involved in keratinocytes response to hyperosmotic shock showing, for the first time, the participation of Low Molecular Weight Protein Tyrosine Phosphatase (LMWPTP) activity in this event. We reported that sorbitol-induced osmotic stress mediates alterations in the phosphorylation of pivotal cytoskeletal proteins, particularly Src and cofilin. Furthermore, an increase in the expression of the phosphorylated form of LMWPTP, which was followed by an augment in its catalytic activity, was observed. Of particular importance, these responses occurred in an intracellular milieu characterized by elevated levels of reduced glutathione (GSH) and increased expression of the antioxidant enzymes glutathione peroxidase and glutathione reductase. Altogether, our results suggest that hyperosmostic stress provides a favorable cellular environment to the activation of LMWPTP, which is associated with increased expression of antioxidant enzymes, high levels of GSH and inhibition of Src kinase. Finally, the real contribution of LMWPTP in the hyperosmotic stress response of keratinocytes was demonstrated through analysis of the effects of ACP1 gene knockdown in stressed and non-stressed cells. LMWPTP knockdown attenuates the effects of sorbitol induced-stress in HaCaT cells, mainly in the status of Src kinase, Rac and STAT5 phosphorylation and activity. These results describe for the first time the participation of LMWPTP in the dynamics of cytoskeleton rearrangement during exposure of human keratinocytes to hyperosmotic shock, which may contribute to cell death. PMID:25781955

  18. Novel Host Proteins and Signaling Pathways in Enteropathogenic E. coli Pathogenesis Identified by Global Phosphoproteome Analysis.

    PubMed

    Scholz, Roland; Imami, Koshi; Scott, Nichollas E; Trimble, William S; Foster, Leonard J; Finlay, B Brett

    2015-07-01

    Enteropathogenic Escherichia coli (EPEC) uses a type III secretion system (T3SS) to directly translocate effector proteins into host cells where they play a pivotal role in subverting host cell signaling needed for disease. However, our knowledge of how EPEC affects host protein phosphorylation is limited to a few individual protein studies. We employed a quantitative proteomics approach to globally map alterations in the host phosphoproteome during EPEC infection. By characterizing host phosphorylation events at various time points throughout infection, we examined how EPEC dynamically impacts the host phosphoproteome over time. This experimental setup also enabled identification of T3SS-dependent and -independent changes in host phosphorylation. Specifically, T3SS-regulated events affected various cellular processes that are known EPEC targets, including cytoskeletal organization, immune signaling, and intracellular trafficking. However, the involvement of phosphorylation in these events has thus far been poorly studied. We confirmed the MAPK family as an established key host player, showed its central role in signal transduction during EPEC infection, and extended the repertoire of known signaling hubs with previously unrecognized proteins, including TPD52, CIN85, EPHA2, and HSP27. We identified altered phosphorylation of known EPEC targets, such as cofilin, where the involvement of phosphorylation has so far been undefined, thus providing novel mechanistic insights into the roles of these proteins in EPEC infection. An overlap of regulated proteins, especially those that are cytoskeleton-associated, was observed when compared with the phosphoproteome of Shigella-infected cells. We determined the biological relevance of the phosphorylation of a novel protein in EPEC pathogenesis, septin-9 (SEPT9). Both siRNA knockdown and a phosphorylation-impaired SEPT9 mutant decreased bacterial adherence and EPEC-mediated cell death. In contrast, a phosphorylation

  19. Filamin-interacting proteins, Cfm1 and Cfm2, are essential for the formation of cartilaginous skeletal elements.

    PubMed

    Mizuhashi, Koji; Kanamoto, Takashi; Moriishi, Takeshi; Muranishi, Yuki; Miyazaki, Toshihiro; Terada, Koji; Omori, Yoshihiro; Ito, Masako; Komori, Toshihisa; Furukawa, Takahisa

    2014-06-01

    Mutations of Filamin genes, which encode actin-binding proteins, cause a wide range of congenital developmental malformations in humans, mainly skeletal abnormalities. However, the molecular mechanisms underlying Filamin functions in skeletal system formation remain elusive. In our screen to identify skeletal development molecules, we found that Cfm (Fam101) genes, Cfm1 (Fam101b) and Cfm2 (Fam101a), are predominantly co-expressed in developing cartilage and intervertebral discs (IVDs). To investigate the functional role of Cfm genes in skeletal development, we generated single knockout mice for Cfm1 and Cfm2, as well as Cfm1/Cfm2 double-knockout (Cfm DKO) mice, by targeted gene disruption. Mice with loss of a single Cfm gene displayed no overt phenotype, whereas Cfm DKO mice showed skeletal malformations including spinal curvatures, vertebral fusions and impairment of bone growth, showing that the phenotypes of Cfm DKO mice resemble those of Filamin B (Flnb)-deficient mice. The number of cartilaginous cells in IVDs is remarkably reduced, and chondrocytes are moderately reduced in Cfm DKO mice. We observed increased apoptosis and decreased proliferation in Cfm DKO cartilaginous cells. In addition to direct interaction between Cfm and Filamin proteins in developing chondrocytes, we showed that Cfm is required for the interaction between Flnb and Smad3, which was reported to regulate Runx2 expression. Furthermore, we found that Cfm DKO primary chondrocytes showed decreased cellular size and fewer actin bundles compared with those of wild-type chondrocytes. These results suggest that Cfms are essential partner molecules of Flnb in regulating differentiation and proliferation of chondryocytes and actin dynamics. PMID:24436304

  20. Mst1 Kinase Regulates the Actin-Bundling Protein L-Plastin To Promote T Cell Migration.

    PubMed

    Xu, Xiaolu; Wang, Xinxin; Todd, Elizabeth M; Jaeger, Emily R; Vella, Jennifer L; Mooren, Olivia L; Feng, Yunfeng; Hu, Jiancheng; Cooper, John A; Morley, Sharon Celeste; Huang, Yina H

    2016-09-01

    Exploring the mechanisms controlling lymphocyte trafficking is essential for understanding the function of the immune system and the pathophysiology of immunodeficiencies. The mammalian Ste20-like kinase 1 (Mst1) has been identified as a critical signaling mediator of T cell migration, and loss of Mst1 results in immunodeficiency disease. Although Mst1 is known to support T cell migration through induction of cell polarization and lamellipodial formation, the downstream effectors of Mst1 are incompletely defined. Mice deficient for the actin-bundling protein L-plastin (LPL) have phenotypes similar to mice lacking Mst1, including decreased T cell polarization, lamellipodial formation, and cell migration. We therefore asked whether LPL functions downstream of Mst1. The regulatory N-terminal domain of LPL contains a consensus Mst1 phosphorylation site at Thr(89) We found that Mst1 can phosphorylate LPL in vitro and that Mst1 can interact with LPL in cells. Removal of the Mst1 phosphorylation site by mutating Thr(89) to Ala impaired localization of LPL to the actin-rich lamellipodia of T cells. Expression of the T89A LPL mutant failed to restore migration of LPL-deficient T cells in vitro. Furthermore, expression of T89A LPL in LPL-deficient hematopoietic cells, using bone marrow chimeras, failed to rescue the phenotype of decreased thymic egress. These results identify LPL as a key effector of Mst1 and establish a novel mechanism linking a signaling intermediate to an actin-binding protein critical to T cell migration. PMID:27465533

  1. Occurrence of caldesmon (a calmodulin-binding protein) in cultured cells: comparison of normal and transformed cells.

    PubMed Central

    Owada, M K; Hakura, A; Iida, K; Yahara, I; Sobue, K; Kakiuchi, S

    1984-01-01

    Caldesmon is a calmodulin-binding and F-actin-binding protein originally purified from chicken gizzard smooth muscle. This protein binds to F-actin filaments in a Ca2+- and calmodulin-dependent "flip-flop" fashion, thereby regulating the function of actin filaments. Here we report that various lines of cultured cells contain a Mr 77,000 protein that specifically reacts with the affinity-purified caldesmon antibody raised against chicken gizzard caldesmon . Among the fibroblast proteins that had been pulse-labeled with [35S]methionine, the Mr 77,000 protein was the only protein band detected on the NaDodSO4 gel that reacted with the anticaldesmon . The subcellular distribution of the Mr 77,000 protein was investigated by the indirect immunofluorescence technique using the anticaldesmon . In all fibroblast cell lines examined, the immunofluorescence localized along the cellular stress fibers and in leading edges of the cell. In Rous sarcoma virus-transformed cells (S7-1), however, the distribution of the fluorescence changed to a diffuse and blurred appearance. These staining patterns of anticaldesmon obtained with the normal and transformed cells coincided with those of antiactin in the corresponding states, strongly suggesting the functional linkage between the Mr 77,000 protein and actin filaments. We propose to refer to this Mr 77,000 protein as caldesmon 77. The cellular level of caldesmon 77 in transformed S7-1 cells decreased to about one-third of that in their normal counterparts (cell line no. 7). Essentially the same result was obtained with normal rat kidney cells infected with the temperature-sensitive transformation mutant Schmidt-Ruppin strain of Rous sarcoma virus (68 N2 clone). The cellular level of caldesmon 77 observed at a permissive temperature (35 degrees C) was about one-third of that at a nonpermissive temperature (38.5 degrees C). These changes of caldesmon 77 in transformed cells may correlate with the loss of Ca2+ regulation in the

  2. Role of the C-terminal Extension of Formin 2 in Its Activation by Spire Protein and Processive Assembly of Actin Filaments.

    PubMed

    Montaville, Pierre; Kühn, Sonja; Compper, Christel; Carlier, Marie-France

    2016-02-12

    Formin 2 (Fmn2), a member of the FMN family of formins, plays an important role in early development. This formin cooperates with profilin and Spire, a WASP homology domain 2 (WH2) repeat protein, to stimulate assembly of a dynamic cytoplasmic actin meshwork that facilitates translocation of the meiotic spindle in asymmetric division of mouse oocytes. The kinase-like non-catalytic domain (KIND) of Spire directly interacts with the C-terminal extension of the formin homology domain 2 (FH2) domain of Fmn2, called FSI. This direct interaction is required for the synergy between the two proteins in actin assembly. We have recently demonstrated how Spire, which caps barbed ends via its WH2 domains, activates Fmn2. Fmn2 by itself associates very poorly to filament barbed ends but is rapidly recruited to Spire-capped barbed ends via the KIND domain, and it subsequently displaces Spire from the barbed end to elicit rapid processive assembly from profilin·actin. Here, we address the mechanism by which Spire and Fmn2 compete at barbed ends and the role of FSI in orchestrating this competition as well as in the processivity of Fmn2. We have combined microcalorimetric, fluorescence, and hydrodynamic binding assays, as well as bulk solution and single filament measurements of actin assembly, to show that removal of FSI converts Fmn2 into a Capping Protein. This activity is mimicked by association of KIND to Fmn2. In addition, FSI binds actin at filament barbed ends as a weak capper and plays a role in displacing the WH2 domains of Spire from actin, thus allowing the association of actin-binding regions of FH2 to the barbed end. PMID:26668326

  3. Reconstitution of actin-based motility of Listeria and Shigella using pure proteins

    NASA Astrophysics Data System (ADS)

    Loisel, Thomas P.; Boujemaa, Rajaa; Pantaloni, Dominique; Carlier, Marie-France

    1999-10-01

    Actin polymerization is essential for cell locomotion and is thought to generate the force responsible for cellular protrusions. The Arp2/3 complex is required to stimulate actin assembly at the leading edge in response to signalling. The bacteria Listeria and Shigella bypass the signalling pathway and harness the Arp2/3 complex to induce actin assembly and to propel themselves in living cells. However, the Arp2/3 complex alone is insufficient to promote movement. Here we have used pure components of the actin cytoskeleton to reconstitute sustained movement in Listeria and Shigella in vitro. Actin-based propulsion is driven by the free energy released by ATP hydrolysis linked to actin polymerization, and does not require myosin. In addition to actin and activated Arp2/3 complex, actin depolymerizing factor (ADF, or cofilin) and capping protein are also required for motility as they maintain a high steady-state level of G-actin, which controls the rate of unidirectional growth of actin filaments at the surface of the bacterium. The movement is more effective when profilin, α-actinin and VASP (for Listeria) are also included. These results have implications for our understanding of the mechanism of actin-based motility in cells.

  4. Cardiac myosin binding protein-C modulates actomyosin binding and kinetics in the in vitro motility assay.

    PubMed

    Saber, Walid; Begin, Kelly J; Warshaw, David M; VanBuren, Peter

    2008-06-01

    The modulatory role of whole cardiac myosin binding protein-C (cMyBP-C) on myosin force and motion generation was assessed in an in vitro motility assay. The presence of cMyBP-C at an approximate molar ratio of cMyBP-C to whole myosin of 1:2, resulted in a 25% reduction in thin filament velocity (P<0.002) with no effect on relative isometric force under maximally activated conditions (pCa 5). Cardiac MyBP-C was capable of inhibiting actin filament velocity in a concentration-dependent manner using either whole myosin, HMM or S1, indicating that the cMyBP-C does not have to bind to myosin LMM or S2 subdomains to exert its effect. The reduction in velocity by cMyBP-C was independent of changes in ionic strength or excess inorganic phosphate. Co-sedimentation experiments demonstrated S1 binding to actin is reduced as a function of cMyBP-C concentration in the presence of ATP. In contrast, S1 avidly bound to actin in the absence of ATP and limited cMyBP-C binding, indicating that cMyBP-C and S1 compete for actin binding in an ATP-dependent fashion. However, based on the relationship between thin filament velocity and filament length, the cMyBP-C induced reduction in velocity was independent of the number of cross-bridges interacting with the thin filament. In conclusion, the effects of cMyBP-C on velocity and force at both maximal and submaximal activation demonstrate that cMyBP-C does not solely act as a tether between the myosin S2 and LMM subdomains but likely affects both the kinetics and recruitment of myosin cross-bridges through its direct interaction with actin and/or myosin head. PMID:18482734

  5. Actin turnover-dependent fast dissociation of capping protein in the dendritic nucleation actin network: evidence of frequent filament severing.

    PubMed

    Miyoshi, Takushi; Tsuji, Takahiro; Higashida, Chiharu; Hertzog, Maud; Fujita, Akiko; Narumiya, Shuh; Scita, Giorgio; Watanabe, Naoki

    2006-12-18

    Actin forms the dendritic nucleation network and undergoes rapid polymerization-depolymerization cycles in lamellipodia. To elucidate the mechanism of actin disassembly, we characterized molecular kinetics of the major filament end-binding proteins Arp2/3 complex and capping protein (CP) using single-molecule speckle microscopy. We have determined the dissociation rates of Arp2/3 and CP as 0.048 and 0.58 s(-1), respectively, in lamellipodia of live XTC fibroblasts. This CP dissociation rate is three orders of magnitude faster than in vitro. CP dissociates slower from actin stress fibers than from the lamellipodial actin network, suggesting that CP dissociation correlates with actin filament dynamics. We found that jasplakinolide, an actin depolymerization inhibitor, rapidly blocked the fast CP dissociation in cells. Consistently, the coexpression of LIM kinase prolonged CP speckle lifetime in lamellipodia. These results suggest that cofilin-mediated actin disassembly triggers CP dissociation from actin filaments. We predict that filament severing and end-to-end annealing might take place fairly frequently in the dendritic nucleation actin arrays. PMID:17178911

  6. Identification of toxicological biomarkers of di(2-ethylhexyl) phthalate in proteins secreted by HepG2 cells using proteomic analysis.

    PubMed

    Choi, Seonyoung; Park, So-Young; Jeong, Ji; Cho, Eunkyung; Phark, Sohee; Lee, Min; Kwak, Dongsub; Lim, Ji-Youn; Jung, Woon-Won; Sul, Donggeun

    2010-05-01

    The effects of di(2-ethylhexyl) phthalate (DEHP) on proteins secreted by HepG2 cells were studied using a proteomic approach. HepG2 cells were exposed to various concentrations of DEHP (0, 2.5, 5, 10, 25, 50, 100, and 250 microM) for 24 or 48 h. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and comet assays were then conducted to determine the cytotoxicity and genotoxicity of DEHP, respectively. The MTT assay showed that 10 microM DEHP was the maximum concentration that did not cause cell death. In addition, the DNA damage in HepG2 cells exposed to DEHP was found to increase in a dose- and time-dependent fashion. Proteomic analysis using two different pI ranges (4-7 and 6-9) and large size 2-DE revealed the presence of 2776 protein spots. A total of 35 (19 up- and 16 down-regulated) proteins were identified as biomarkers of DEHP by ESI-MS/MS. Several differentiated protein groups were also found. Proteins involved in apoptosis, transportation, signaling, energy metabolism, and cell structure and motility were found to be up- or down-regulated. Among these, the identities of cystatin C, Rho GDP inhibitor, retinol binding protein 4, gelsolin, DEK protein, Raf kinase inhibitory protein, triose phosphate isomerase, cofilin-1, and haptoglobin-related protein were confirmed by Western blot assay. Therefore, these proteins could be used as potential biomarkers of DEHP and human disease associated with DEHP. PMID:20198640

  7. Computational Study of the Binding Mechanism of Actin-Depolymerizing Factor 1 with Actin in Arabidopsis thaliana

    PubMed Central

    Wang, Xue; Dong, Chun-Hai; Yang, Jian Ming; Yao, Xiao Jun

    2016-01-01

    Actin is a highly conserved protein. It plays important roles in cellular function and exists either in the monomeric (G-actin) or polymeric form (F-actin). Members of the actin-depolymerizing factor (ADF)/cofilin protein family bind to both G-actin and F-actin and play vital roles in actin dynamics by manipulating the rates of filament polymerization and depolymerization. It has been reported that the S6D and R98A/K100A mutants of actin-depolymerizing factor 1 (ADF1) in Arabidopsis thaliana decreased the binding affinity of ADF for the actin monomer. To investigate the binding mechanism and dynamic behavior of the ADF1–actin complex, we constructed a homology model of the AtADF1–actin complex based on the crystal structure of AtADF1 and the twinfilin C-terminal ADF-H domain in a complex with a mouse actin monomer. The model was then refined for subsequent molecular dynamics simulations. Increased binding energy of the mutated system was observed using the Molecular Mechanics Generalized Born Surface Area and Poisson–Boltzmann Surface Area (MM-GB/PBSA) methods. To determine the residues that make decisive contributions to the ADF1 actin-binding affinity, per-residue decomposition and computational alanine scanning analyses were performed, which provided more detailed information on the binding mechanism. Root-mean-square fluctuation and principal component analyses confirmed that the S6D and R98A/K100A mutants induced an increased conformational flexibility. The comprehensive molecular insight gained from this study is of great importance for understanding the binding mechanism of ADF1 and G-actin. PMID:27414648

  8. Computational Study of the Binding Mechanism of Actin-Depolymerizing Factor 1 with Actin in Arabidopsis thaliana.

    PubMed

    Du, Juan; Wang, Xue; Dong, Chun-Hai; Yang, Jian Ming; Yao, Xiao Jun

    2016-01-01

    Actin is a highly conserved protein. It plays important roles in cellular function and exists either in the monomeric (G-actin) or polymeric form (F-actin). Members of the actin-depolymerizing factor (ADF)/cofilin protein family bind to both G-actin and F-actin and play vital roles in actin dynamics by manipulating the rates of filament polymerization and depolymerization. It has been reported that the S6D and R98A/K100A mutants of actin-depolymerizing factor 1 (ADF1) in Arabidopsis thaliana decreased the binding affinity of ADF for the actin monomer. To investigate the binding mechanism and dynamic behavior of the ADF1-actin complex, we constructed a homology model of the AtADF1-actin complex based on the crystal structure of AtADF1 and the twinfilin C-terminal ADF-H domain in a complex with a mouse actin monomer. The model was then refined for subsequent molecular dynamics simulations. Increased binding energy of the mutated system was observed using the Molecular Mechanics Generalized Born Surface Area and Poisson-Boltzmann Surface Area (MM-GB/PBSA) methods. To determine the residues that make decisive contributions to the ADF1 actin-binding affinity, per-residue decomposition and computational alanine scanning analyses were performed, which provided more detailed information on the binding mechanism. Root-mean-square fluctuation and principal component analyses confirmed that the S6D and R98A/K100A mutants induced an increased conformational flexibility. The comprehensive molecular insight gained from this study is of great importance for understanding the binding mechanism of ADF1 and G-actin. PMID:27414648

  9. Single Molecule Science for Personalized Nanomedicine: Atomic Force Microscopy of Biopolymer-Protein Interactions

    NASA Astrophysics Data System (ADS)

    Hsueh, Carlin

    Nanotechnology has a unique and relatively untapped utility in the fields of medicine and dentistry at the level of single-biopolymer and -molecule diagnostics. In recent years atomic force microscopy (AFM) has garnered much interest due to its ability to obtain atomic-resolution of molecular structures and probe biophysical behaviors of biopolymers and proteins in a variety of biologically significant environments. The work presented in this thesis focuses on the nanoscale manipulation and observation of biopolymers to develop an innovative technology for personalized medicine while understanding complex biological systems. These studies described here primarily use AFM to observe biopolymer interactions with proteins and its surroundings with unprecedented resolution, providing a better understanding of these systems and interactions at the nanoscale. Transcriptional profiling, the measure of messenger RNA (mRNA) abundance in a single cell, is a powerful technique that detects "behavior" or "symptoms" at the tissue and cellular level. We have sought to develop an alternative approach, using our expertise in AFM and single molecule nanotechnology, to achieve a cost-effective high throughput method for sensitive detection and profiling of subtle changes in transcript abundance. The technique does not require amplification of the mRNA sample because the AFM provides three-dimensional views of molecules with unprecedented resolution, requires minimal sample preparation, and utilizes a simple tagging chemistry on cDNA molecules. AFM images showed collagen polymers in teeth and of Drebrin-A remodeling of filamentous actin structure and mechanics. AFM was used to image collagen on exposed dentine tubules and confirmed tubule occlusion with a desensitizing prophylaxis paste by Colgate-Palmolive. The AFM also superseded other microscopy tools in resolving F-actin helix remodeling and possible cooperative binding by a neuronal actin binding protein---Drebrin-A, an

  10. PLPP/CIN regulates bidirectional synaptic plasticity via GluN2A interaction with postsynaptic proteins

    PubMed Central

    Kim, Ji-Eun; Kim, Yeon-Joo; Lee, Duk-Shin; Kim, Ji Yang; Ko, Ah-Reum; Hyun, Hye-Won; Kim, Min Ju; Kang, Tae-Cheon

    2016-01-01

    Dendritic spines are dynamic structures whose efficacies and morphologies are modulated by activity-dependent synaptic plasticity. The actin cytoskeleton plays an important role in stabilization and structural modification of spines. However, the regulatory mechanism by which it alters the plasticity threshold remains elusive. Here, we demonstrate the role of pyridoxal-5′-phosphate phosphatase/chronophin (PLPP/CIN), one of the cofilin-mediated F-actin regulators, in modulating synaptic plasticity in vivo. PLPP/CIN transgenic (Tg) mice had immature spines with small heads, while PLPP/CIN knockout (KO) mice had gigantic spines. Furthermore, PLPP/CIN Tg mice exhibited enhanced synaptic plasticity, but KO mice showed abnormal synaptic plasticity. The PLPP/CIN-induced alterations in synaptic plasticity were consistent with the acquisition and the recall capacity of spatial learning. PLPP/CIN also enhanced N-methyl-D-aspartate receptor (GluN) functionality by regulating the coupling of GluN2A with interacting proteins, particularly postsynaptic density-95 (PSD95). Therefore, these results suggest that PLPP/CIN may be an important factor for regulating the plasticity threshold. PMID:27212638

  11. The M. tuberculosis HAD phosphatase (Rv3042c) interacts with host proteins and is inhibited by Clofazimine.

    PubMed

    Shree, Sonal; Singh, Abhishek Kumar; Saxena, Richa; Kumar, Harish; Agarwal, Aparna; Sharma, Vijay Kumar; Srivastava, Kanchan; Srivastava, Kishore Kumar; Sanyal, Sabyasachi; Ramachandran, Ravishankar

    2016-09-01

    Mycobacterium tuberculosis codes for a HAD-phosphatase, Rv3042c (MtSerB2), that has earlier been characterized as a metabolic enzyme. Here we demonstrate that MtSerB2 is secreted into the cytosol of infected macrophages and is found in bronchoalveolar lavage samples of tuberculosis patients. MtSerB2 induces significant cytoskeleton rearrangements through cofilin activation and affects the expression of genes that regulate actin dynamics. It specifically interacts with HSP90, HSP70 and HSP27 that block apoptotic pathways and not with other HSPs. It actively dephosphorylates MAPK-p38 and NF-kappa B p65 that play crucial roles in inflammatory and immune responses. This in turn leads to down-regulation of Interleukin 8, a chemotactic and inflammatory cytokine. Finally, during evaluation of inhibitors against MtSerB2 we found that Clofazimine, a drug being evaluated for XDR and MDR tuberculosis, inhibits MtSerB2 phosphatase activity and reverses the above effects and interactions with host proteins. Overall, the study identifies that MtSerB2 has new functions that might help the pathogen to evade the host's immune response. PMID:26984196

  12. Changes in cell adhesivity and cytoskeleton-related proteins during imatinib-induced apoptosis of leukemic JURL-MK1 cells.

    PubMed

    Kuželová, K; Pluskalová, M; Grebeňová, D; Pavlásková, K; Halada, P; Hrkal, Z

    2010-12-15

    The fusion protein Bcr-Abl, which is the molecular cause of chronic myelogenous leukemia (CML) interacts in multiple points with signaling pathways regulating the cellular adhesivity and cytoskeleton architecture and dynamics. We explored the effects of imatinib mesylate, an inhibitor of Bcr-Abl protein used in front-line CML therapy, on the adhesivity of JURL-MK1 cells to fibronectin and searched for underlying changes in the cell proteome. As imatinib induces apoptosis of JURL-MK1 cells, we used three different caspase inhibitors to discriminate between direct consequences of Bcr-Abl inhibition and secondary changes related to the apoptosis. Imatinib treatment caused a transient increase in JURL-MK1 cell adhesivity to fibronectin, possibly due to the switch off of Bcr-Abl activity. Subsequently, we observed a number of changes including a decrease in cell adhesivity, F-actin decomposition, reduction of integrin β1, CD44, and paxillin expression levels and a marked increase in cofilin phophorylation at Ser3. These events were generally related to the proceeding apoptosis but they differed in their sensitivity to the individual caspase inhibitors. PMID:20830748

  13. A nanobody targeting the F-actin capping protein CapG restrains breast cancer metastasis

    PubMed Central

    2013-01-01

    Introduction Aberrant turnover of the actin cytoskeleton is intimately associated with cancer cell migration and invasion. Frequently however, evidence is circumstantial, and a reliable assessment of the therapeutic significance of a gene product is offset by lack of inhibitors that target biologic properties of a protein, as most conventional drugs do, instead of the corresponding gene. Proteomic studies have demonstrated overexpression of CapG, a constituent of the actin cytoskeleton, in breast cancer. Indirect evidence suggests that CapG is involved in tumor cell dissemination and metastasis. In this study, we used llama-derived CapG single-domain antibodies or nanobodies in a breast cancer metastasis model to address whether inhibition of CapG activity holds therapeutic merit. Methods We raised single-domain antibodies (nanobodies) against human CapG and used these as intrabodies (immunomodulation) after lentiviral transduction of breast cancer cells. Functional characterization of nanobodies was performed to identify which biochemical properties of CapG are perturbed. Orthotopic and tail vein in vivo models of metastasis in nude mice were used to assess cancer cell spreading. Results With G-actin and F-actin binding assays, we identified a CapG nanobody that binds with nanomolar affinity to the first CapG domain. Consequently, CapG interaction with actin monomers or actin filaments is blocked. Intracellular delocalization experiments demonstrated that the nanobody interacts with CapG in the cytoplasmic environment. Expression of the nanobody in breast cancer cells restrained cell migration and Matrigel invasion. Notably, the nanobody prevented formation of lung metastatic lesions in orthotopic xenograft and tail-vein models of metastasis in immunodeficient mice. We showed that CapG nanobodies can be delivered into cancer cells by using bacteria harboring a type III protein secretion system (T3SS). Conclusions CapG inhibition strongly reduces breast cancer

  14. Epstein-Barr virus infection induces expression in B lymphocytes of a novel gene encoding an evolutionarily conserved 55-kilodalton actin-bundling protein.

    PubMed

    Mosialos, G; Yamashiro, S; Baughman, R W; Matsudaira, P; Vara, L; Matsumura, F; Kieff, E; Birkenbach, M

    1994-11-01

    A novel human mRNA whose expression is induced over 200-fold in B lymphocytes by latent Epstein-Barr virus (EBV) infection was reverse transcribed, cloned, and sequenced. The mRNA is predicted to encode a protein containing four peptides which precisely match amino acid sequences from a previously identified 55-kDa actin-bundling protein, p55. In vitro translation of the cDNA results in a 55-kDa protein which binds to actin filaments in the presence of purified p55 from HeLa cells. The p55 mRNA is undetectable in non-EBV-infected B- and T-cell lines or in a myelomonocytic cell line (U937). Newly infected primary human B lymphocytes, EBV-transformed B-cell lines, latently infected Burkitt tumor cells expressing EBNA2 and LMP1, a chronic myelogenous leukemia cell line (K562), and an osteosarcoma cell line (TK143) contain high levels of p55 mRNA or protein. In EBV-transformed B cells, p55 localizes to perinuclear cytoplasm and to cell surface processes that resemble filopodia. The p55 mRNA is detected at high levels in spleen and brain tissues, at moderate levels in lung and placenta tissues, and at low levels in skeletal muscle, liver, and tonsil tissues and is undetectable in heart, kidney, pancreas, and bone marrow tissues. Immunohistochemical staining of human brain tissue demonstrates p55 localization to the perinuclear cytoplasm and dendritic processes of many, but not all, types of cortical or cerebellar neurons, to glial cells, and to capillary endothelial cells. In cultured primary rat neurons, p55 is distributed throughout the perinuclear cytoplasm and in subcortical filamentous structures of dendrites and growth cones. p55 is highly evolutionarily conserved since it shows 40% amino acid sequence identity to the Drosophila singed gene product and 37% identity to fascin, an echinoderm actin-bundling protein. The evolutionary conservation of p55 and its lack of extensive homology to other actin-binding proteins suggest that p55 has specific microfilament

  15. The Cyclase-Associated Protein Cap1 Is Important for Proper Regulation of Infection-Related Morphogenesis in Magnaporthe oryzae

    PubMed Central

    Zhou, Xiaoying; Zhang, Haifeng; Li, Guotian; Shaw, Brian; Xu, Jin-Rong

    2012-01-01

    Surface recognition and penetration are critical steps in the infection cycle of many plant pathogenic fungi. In Magnaporthe oryzae, cAMP signaling is involved in surface recognition and pathogenesis. Deletion of the MAC1 adenylate cyclase gene affected appressorium formation and plant infection. In this study, we used the affinity purification approach to identify proteins that are associated with Mac1 in vivo. One of the Mac1-interacting proteins is the adenylate cyclase-associated protein named Cap1. CAP genes are well-conserved in phytopathogenic fungi but none of them have been functionally characterized. Deletion of CAP1 blocked the effects of a dominant RAS2 allele and resulted in defects in invasive growth and a reduced intracellular cAMP level. The Δcap1 mutant was defective in germ tube growth, appressorium formation, and formation of typical blast lesions. Cap1-GFP had an actin-like localization pattern, localizing to the apical regions in vegetative hyphae, at the periphery of developing appressoria, and in circular structures at the base of mature appressoria. Interestingly, Cap1, similar to LifeAct, did not localize to the apical regions in invasive hyphae, suggesting that the apical actin cytoskeleton differs between vegetative and invasive hyphae. Domain deletion analysis indicated that the proline-rich region P2 but not the actin-binding domain (AB) of Cap1 was responsible for its subcellular localization. Nevertheless, the AB domain of Cap1 must be important for its function because CAP1ΔAB only partially rescued the Δcap1 mutant. Furthermore, exogenous cAMP induced the formation of appressorium-like structures in non-germinated conidia in CAP1ΔAB transformants. This novel observation suggested that AB domain deletion may result in overstimulation of appressorium formation by cAMP treatment. Overall, our results indicated that CAP1 is important for the activation of adenylate cyclase, appressorium morphogenesis, and plant infection in M

  16. 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.

  17. 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.

  18. The Association of Myosin IB with Actin Waves in Dictyostelium Requires Both the Plasma Membrane-Binding Site and Actin-Binding Region in the Myosin Tail

    PubMed Central

    Brzeska, Hanna; Pridham, Kevin; Chery, Godefroy; Titus, Margaret A.; Korn, Edward D.

    2014-01-01

    F-actin structures and their distribution are important determinants of the dynamic shapes and functions of eukaryotic cells. Actin waves are F-actin formations that move along the ventral cell membrane driven by actin polymerization. Dictyostelium myosin IB is associated with actin waves but its role in the wave is unknown. Myosin IB is a monomeric, non-filamentous myosin with a globular head that binds to F-actin and has motor activity, and a non-helical tail comprising a basic region, a glycine-proline-glutamine-rich region and an SH3-domain. The basic region binds to acidic phospholipids in the plasma membrane through a short basic-hydrophobic site and the Gly-Pro-Gln region binds F-actin. In the current work we found that both the basic-hydrophobic site in the basic region and the Gly-Pro-Gln region of the tail are required for the association of myosin IB with actin waves. This is the first evidence that the Gly-Pro-Gln region is required for localization of myosin IB to a specific actin structure in situ. The head is not required for myosin IB association with actin waves but binding of the head to F-actin strengthens the association of myosin IB with waves and stabilizes waves. Neither the SH3-domain nor motor activity is required for association of myosin IB with actin waves. We conclude that myosin IB contributes to anchoring actin waves to the plasma membranes by binding of the basic-hydrophobic site to acidic phospholipids in the plasma membrane and binding of the Gly-Pro-Gln region to F-actin in the wave. PMID:24747353

  19. 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 ...

  20. Proteins implicated in the increase of adhesivity induced by suberoylanilide hydroxamic acid in leukemic cells.

    PubMed

    Grebeňová, D; Röselová, P; Pluskalová, M; Halada, P; Rösel, D; Suttnar, J; Brodská, B; Otevřelová, P; Kuželová, K

    2012-12-21

    We have previously shown that suberoylanilide hydroxamic acid (SAHA) treatment increases the adhesivity of leukemic cells to fibronectin at clinically relevant concentrations. Now, we present the results of the proteomic analysis of SAHA effects on leukemic cell lines using 2-DE and ProteomLab PF2D system. Histone acetylation at all studied acetylation sites reached the maximal level after 5 to 10 h of SAHA treatment. No difference in histone acetylation between subtoxic and toxic SAHA doses was observed. SAHA treatment induced cofilin phosphorylation at Ser3, an increase in vimentin and paxillin expression and a decrease in stathmin expression as confirmed by western-blotting and immunofluorescence microscopy. The interaction of cofilin with 14-3-3 epsilon was documented using both Duolink system and coimmunoprecipitation. However, this interaction was independent of cofilin Ser3 phosphorylation and the amount of 14-3-3-ε-bound cofilin did not rise following SAHA treatment. SAHA-induced increase in the cell adhesivity was associated with an increase in PAK phosphorylation in CML-T1 cells and was abrogated by simultaneous treatment with IPA-3, a PAK inhibitor. The effects of SAHA on JURL-MK1 cells were similar to those of other histone deacetylase inhibitors, tubastatin A and sodium butyrate. The proteome analysis also revealed several potential non-histone targets of histone deacetylases. PMID:23022583

  1. 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

  2. The Tandem PH Domain-Containing Protein 2 (TAPP2) Regulates Chemokine-Induced Cytoskeletal Reorganization and Malignant B Cell Migration

    PubMed Central

    Li, Hongzhao; Hou, Sen; Wu, Xun; Nandagopal, Saravanan; Lin, Francis; Kung, Sam; Marshall, Aaron James

    2013-01-01

    The intracellular signaling processes controlling malignant B cell migration and tissue localization remain largely undefined. Tandem PH domain-containing proteins TAPP1 and TAPP2 are adaptor proteins that specifically bind to phosphatidylinositol-3,4-bisphosphate, or PI(3,4)P2, a product of phosphoinositide 3-kinases (PI3K). While PI3K enzymes have a number of functions in cell biology, including cell migration, the functions of PI(3,4)P2 and its binding proteins are not well understood. Previously we found that TAPP2 is highly expressed in primary leukemic B cells that have strong migratory capacity. Here we find that SDF-1-dependent migration of human malignant B cells requires both PI3K signaling and TAPP2. Migration in a transwell assay is significantly impaired by pan-PI3K and isoform-selective PI3K inhibitors, or by TAPP2 shRNA knockdown (KD). Strikingly, TAPP2 KD in combination with PI3K inhibitor treatment nearly abolished the migration response, suggesting that TAPP2 may contribute some functions independent of the PI3K pathway. In microfluidic chamber cell tracking assays, TAPP2 KD cells show reduction in percentage of migrating cells, migration velocity and directionality. TAPP2 KD led to alterations in chemokine-induced rearrangement of the actin cytoskeleton and failure to form polarized morphology. TAPP2 co-localized with the stable F-actin-binding protein utrophin, with both molecules reciprocally localizing against F-actin accumulated at the leading edge upon SDF-1 stimulation. In TAPP2 KD cells, Rac was over-activated and localized to multiple membrane protrusions, suggesting that TAPP2 may act in concert with utrophin and stable F-actin to spatially restrict Rac activation and reduce formation of multiple membrane protrusions. TAPP2 function in cell migration is also apparent in the more complex context of B cell migration into stromal cell layers – a process that is only partially dependent on PI3K and SDF-1. In summary, this study identified

  3. Dynamic Regulation of a Cell Adhesion Protein Complex Including CADM1 by Combinatorial Analysis of FRAP with Exponential Curve-Fitting

    PubMed Central

    Sakurai-Yageta, Mika; Maruyama, Tomoko; Suzuki, Takashi; Ichikawa, Kazuhisa; Murakami, Yoshinori

    2015-01-01

    Protein components of cell adhesion machinery show continuous renewal even in the static state of epithelial cells and participate in the formation and maintenance of normal epithelial architecture and tumor suppression. CADM1 is a tumor suppressor belonging to the immunoglobulin superfamily of cell adhesion molecule and forms a cell adhesion complex with an actin-binding protein, 4.1B, and a scaffold protein, MPP3, in the cytoplasm. Here, we investigate dynamic regulation of the CADM1-4.1B-MPP3 complex in mature cell adhesion by fluorescence recovery after photobleaching (FRAP) analysis. Traditional FRAP analysis were performed for relatively short period of around 10min. Here, thanks to recent advances in the sensitive laser detector systems, we examine FRAP of CADM1 complex for longer period of 60 min and analyze the recovery with exponential curve-fitting to distinguish the fractions with different diffusion constants. This approach reveals that the fluorescence recovery of CADM1 is fitted to a single exponential function with a time constant (τ) of approximately 16 min, whereas 4.1B and MPP3 are fitted to a double exponential function with two τs of approximately 40-60 sec and 16 min. The longer τ is similar to that of CADM1, suggesting that 4.1B and MPP3 have two distinct fractions, one forming a complex with CADM1 and the other present as a free pool. Fluorescence loss in photobleaching analysis supports the presence of a free pool of these proteins near the plasma membrane. Furthermore, double exponential fitting makes it possible to estimate the ratio of 4.1B and MPP3 present as a free pool and as a complex with CADM1 as approximately 3:2 and 3:1, respectively. Our analyses reveal a central role of CADM1 in stabilizing the complex with 4.1B and MPP3 and provide insight in the dynamics of adhesion complex formation. PMID:25780926

  4. Overexpression of Isoforms of Nitric Oxide Synthase 1 Adaptor Protein, Encoded by a Risk Gene for Schizophrenia, Alters Actin Dynamics and Synaptic Function

    PubMed Central

    Hernandez, Kristina; Swiatkowski, Przemyslaw; Patel, Mihir V.; Liang, Chen; Dudzinski, Natasha R.; Brzustowicz, Linda M.; Firestein, Bonnie L.

    2016-01-01

    Proper communication between neurons depends upon appropriate patterning of dendrites and correct distribution and structure of spines. Schizophrenia is a neuropsychiatric disorder characterized by alterations in dendrite branching and spine density. Nitric oxide synthase 1 adaptor protein (NOS1AP), a risk gene for schizophrenia, encodes proteins that are upregulated in the dorsolateral prefrontal cortex (DLPFC) of individuals with schizophrenia. To elucidate the effects of NOS1AP overexpression observed in individuals with schizophrenia, we investigated changes in actin dynamics and spine development when a long (NOS1AP-L) or short (NOS1AP-S) isoform of NOS1AP is overexpressed. Increased NOS1AP-L protein promotes the formation of immature spines when overexpressed in rat cortical neurons from day in vitro (DIV) 14 to DIV 17 and reduces the amplitude of miniature excitatory postsynaptic currents (mEPSCs). In contrast, increased NOS1AP-S protein increases the rate of actin polymerization and the number of immature and mature spines, which may be attributed to a decrease in total Rac1 expression and a reduction in the levels of active cofilin. The increase in the number of mature spines by overexpression of NOS1AP-S is accompanied by an increase in the frequency of mEPSCs. Our findings show that overexpression of NOS1AP-L or NOS1AP-S alters the actin cytoskeleton and synaptic function. However, the mechanisms by which these isoforms induce these changes are distinct. These results are important for understanding how increased expression of NOS1AP isoforms can influence spine development and synaptic function. PMID:26869880

  5. Overexpression of Isoforms of Nitric Oxide Synthase 1 Adaptor Protein, Encoded by a Risk Gene for Schizophrenia, Alters Actin Dynamics and Synaptic Function.

    PubMed

    Hernandez, Kristina; Swiatkowski, Przemyslaw; Patel, Mihir V; Liang, Chen; Dudzinski, Natasha R; Brzustowicz, Linda M; Firestein, Bonnie L

    2016-01-01

    Proper communication between neurons depends upon appropriate patterning of dendrites and correct distribution and structure of spines. Schizophrenia is a neuropsychiatric disorder characterized by alterations in dendrite branching and spine density. Nitric oxide synthase 1 adaptor protein (NOS1AP), a risk gene for schizophrenia, encodes proteins that are upregulated in the dorsolateral prefrontal cortex (DLPFC) of individuals with schizophrenia. To elucidate the effects of NOS1AP overexpression observed in individuals with schizophrenia, we investigated changes in actin dynamics and spine development when a long (NOS1AP-L) or short (NOS1AP-S) isoform of NOS1AP is overexpressed. Increased NOS1AP-L protein promotes the formation of immature spines when overexpressed in rat cortical neurons from day in vitro (DIV) 14 to DIV 17 and reduces the amplitude of miniature excitatory postsynaptic currents (mEPSCs). In contrast, increased NOS1AP-S protein increases the rate of actin polymerization and the number of immature and mature spines, which may be attributed to a decrease in total Rac1 expression and a reduction in the levels of active cofilin. The increase in the number of mature spines by overexpression of NOS1AP-S is accompanied by an increase in the frequency of mEPSCs. Our findings show that overexpression of NOS1AP-L or NOS1AP-S alters the actin cytoskeleton and synaptic function. However, the mechanisms by which these isoforms induce these changes are distinct. These results are important for understanding how increased expression of NOS1AP isoforms can influence spine development and synaptic function. PMID:26869880

  6. Two domains of the epidermal growth factor receptor are involved in cytoskeletal interactions

    SciTech Connect

    Song Wei; Wu Jing; Ge Gaoxiang; Lin Qishui

    2008-06-13

    Epidermal growth factor receptor can interact directly with F-actin through an actin-binding domain. In the present study, a mutant EGFR, lacking a previously identified actin-binding domain (ABD 1), was still able to bind elements of the cytoskeleton. A second EGFR actin-binding domain (ABD 2) was identified in the region of the receptor that includes Tyr-1148 by a yeast two-hybrid assay. GST fusion proteins comprising ABD 1 or ABD 2 bound actin in vitro and competed for actin-binding with the full-length EGFR. EGFR binding to actin was also studied in intact cells using fluorescence resonance energy transfer (FRET). The localization of the EGFR/actin-binding complex changed after EGF stimulation. Fusion proteins containing mutations in ABD1 or ABD2 did not display a FRET signal. The results lead to the conclusion that the interaction between ABD1 and ABD2 and actin during EGF-induced signal transduction, and thus between EGFR and actin, are important in cell activation.

  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. 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 ...

  10. 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 ...

  11. 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.

  12. 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…

  13. 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

  14. 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.

  15. 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

  16. 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 ...

  17. Immunofluorescence localization of the unconventional myosin, Myo2p, and the putative kinesin-related protein, Smy1p, to the same regions of polarized growth in Saccharomyces cerevisiae

    PubMed Central

    1994-01-01

    that the myo2 mutant can recover from osmotic shift (unlike actin mutants; Novick, P., and D. Botstein. 1985. Cell. 40:405-416). We have also determined that the myo2-66 allele encodes a Lys instead of a Glu at position 511, which lies at an actin-binding face in the motor domain. PMID:8188749

  18. Neospora caninum: Cloning and expression of a gene coding for cytokine-inducing Neospora caninum profilin

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Profilins are actin-binding proteins that in T. gondii stimulate innate immunity in mice by binding Toll-like receptors (TLR) on dendritic cells (DC) leading to release of inflammatory cytokines, primarily IL-12 and IFN-'. The purpose of the present study was to characterize Neospora caninum profil...

  19. Molecular and cellular characterization of the tomato pollen profilin, LePro1

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Profilin is an actin-binding protein involved in the dynamic turnover and restructuring of the actin cytoskeleton in all eukaryotic cells. We previously cloned a profilin gene, designated as LePro1 from tomato pollen. To investigate its biological role, in the present study, We investigated the tem...

  20. Reduced dimensionality tailored HN(C)N experiments for facile backbone resonance assignment of proteins through unambiguous identification of sequential HSQC peaks

    NASA Astrophysics Data System (ADS)

    Kumar, Dinesh

    2013-12-01

    Two novel reduced dimensionality (RD) tailored HN(C)N [S.C. Panchal, N.S. Bhavesh, R.V. Hosur, Improved 3D triple resonance experiments, HNN and HN(C)N, for HN and 15N sequential correlations in (13C, 15N) labeled proteins: application to unfolded proteins, J. Biomol. NMR 20 (2001) 135-147] experiments are proposed to facilitate the backbone resonance assignment of proteins both in terms of its accuracy and speed. These experiments - referred here as (4,3)D-hNCOcaNH and (4,3)D-hNcoCANH - exploit the linear combination of backbone 15N and 13C‧/13Cα chemical shifts simultaneously to achieve higher peak dispersion and randomness along their respective F1 dimensions. Simply, this has been achieved by modulating the backbone 15N(i) chemical shifts with that of 13C‧ (i - 1)/13Cα (i - 1) spins following the established reduced dimensionality NMR approach [T. Szyperski, D.C. Yeh, D.K. Sukumaran, H.N. Moseley, G.T. Montelione, Reduced-dimensionality NMR spectroscopy for high-throughput protein resonance assignment, Proc. Natl. Acad. Sci. USA 99 (2002) 8009-8014]. Though the modification is simple it has resulted an ingenious improvement of HN(C)N both in terms of peak dispersion and easiness of establishing the sequential connectivities. The increased dispersion along F1 dimension solves two purposes here: (i) resolves the ambiguities arising because of degenerate 15N chemical shifts and (ii) reduces the signal overlap in F2(15N)-F3(1H) planes (an important requisite in HN(C)N based assignment protocol for facile and unambiguous identification of sequentially connected HSQC peaks). The performance of both these experiments and the assignment protocol has been demonstrated using bovine apo Calbindin-d9k (75 aa) and urea denatured UNC60B (a 152 amino acid ADF/cofilin family protein of Caenorhabditis elegans), as representatives of folded and unfolded protein systems, respectively.

  1. Reduced dimensionality tailored HN(C)N experiments for facile backbone resonance assignment of proteins through unambiguous identification of sequential HSQC peaks.

    PubMed

    Kumar, Dinesh

    2013-12-01

    Two novel reduced dimensionality (RD) tailored HN(C)N [S.C. Panchal, N.S. Bhavesh, R.V. Hosur, Improved 3D triple resonance experiments, HNN and HN(C)N, for HN and 15N sequential correlations in (13C, 15N) labeled proteins: application to unfolded proteins, J. Biomol. NMR 20 (2001) 135-147] experiments are proposed to facilitate the backbone resonance assignment of proteins both in terms of its accuracy and speed. These experiments - referred here as (4,3)D-hNCOcaNH and (4,3)D-hNcoCANH - exploit the linear combination of backbone (15)N and (13)C'/(13)C(α) chemical shifts simultaneously to achieve higher peak dispersion and randomness along their respective F1 dimensions. Simply, this has been achieved by modulating the backbone (15)N(i) chemical shifts with that of (13)C' (i-1)/(13)C(α) (i-1) spins following the established reduced dimensionality NMR approach [T. Szyperski, D.C. Yeh, D.K. Sukumaran, H.N. Moseley, G.T. Montelione, Reduced-dimensionality NMR spectroscopy for high-throughput protein resonance assignment, Proc. Natl. Acad. Sci. USA 99 (2002) 8009-8014]. Though the modification is simple it has resulted an ingenious improvement of HN(C)N both in terms of peak dispersion and easiness of establishing the sequential connectivities. The increased dispersion along F1 dimension solves two purposes here: (i) resolves the ambiguities arising because of degenerate (15)N chemical shifts and (ii) reduces the signal overlap in F2((15)N)-F3((1)H) planes (an important requisite in HN(C)N based assignment protocol for facile and unambiguous identification of sequentially connected HSQC peaks). The performance of both these experiments and the assignment protocol has been demonstrated using bovine apo Calbindin-d9k (75 aa) and urea denatured UNC60B (a 152 amino acid ADF/cofilin family protein of Caenorhabditis elegans), as representatives of folded and unfolded protein systems, respectively. PMID:24161682

  2. 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

  3. 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.

  4. 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

  5. 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...

  6. Probing the Flexibility of Tropomyosin and Its Binding to Filamentous Actin Using Molecular Dynamics Simulations

    PubMed Central

    Zheng, Wenjun; Barua, Bipasha; Hitchcock-DeGregori, Sarah E.

    2013-01-01

    Tropomyosin (Tm) is a coiled-coil protein that binds to filamentous actin (F-actin) and regulates its interactions with actin-binding proteins like myosin by moving between three positions on F-actin (the blocked, closed, and open positions). To elucidate the molecular details of Tm flexibility in relation to its binding to F-actin, we conducted extensive molecular dynamics simulations for both Tm alone and Tm-F-actin complex in the presence of explicit solvent (total simulation time >400 ns). Based on the simulations, we systematically analyzed the local flexibility of the Tm coiled coil using multiple parameters. We found a good correlation between the regions with high local flexibility and a number of destabilizing regions in Tm, including six clusters of core alanines. Despite the stabilization by F-actin binding, the distribution of local flexibility in Tm is largely unchanged in the absence and presence of F-actin. Our simulations showed variable fluctuations of individual Tm periods from the closed position toward the open position. In addition, we performed Tm-F-actin binding calculations based on the simulation trajectories, which support the importance of Tm flexibility to Tm-F-actin binding. We identified key residues of Tm involved in its dynamic interactions with F-actin, many of which have been found in recent mutational studies to be functionally important, and the rest of which will make promising targets for future mutational experiments. PMID:24138864

  7. 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

  8. Caspase-11 and caspase-1 differentially modulate actin polymerization via RhoA and Slingshot proteins to promote bacterial clearance

    PubMed Central

    Caution, Kyle; Gavrilin, Mikhail A.; Tazi, Mia; Kanneganti, Apurva; Layman, Daniel; Hoque, Sheshadri; Krause, Kathrin; Amer, Amal O.

    2015-01-01

    Inflammasomes are multiprotein complexes that include members of the NOD-like receptor family and caspase-1. Caspase-1 is required for the fusion of the Legionella vacuole with lysosomes. Caspase-11, independently of the inflammasome, also promotes phagolysosomal fusion. However, it is unclear how these proteases alter intracellular trafficking. Here, we show that caspase-11 and caspase-1 function in opposing manners to phosphorylate and dephosphorylate cofilin, respectively upon infection with Legionella. Caspase-11 targets cofilin via the RhoA GTPase, whereas caspase-1 engages the Slingshot phosphatase. The absence of either caspase-11 or caspase-1 maintains actin in the polymerized or depolymerized form, respectively and averts the fusion of pathogen-containing vacuoles with lysosomes. Therefore, caspase-11 and caspase-1 converge on the actin machinery with opposing effects to promote vesicular trafficking. PMID:26686473

  9. A short splicing isoform of afadin suppresses the cortical axon branching in a dominant-negative manner.

    PubMed

    Umeda, Kentaro; Iwasawa, Nariaki; Negishi, Manabu; Oinuma, Izumi

    2015-05-15

    Precise wiring patterns of axons are among the remarkable features of neuronal circuit formation, and establishment of the proper neuronal network requires control of outgrowth, branching, and guidance of axons. R-Ras is a Ras-family small GTPase that has essential roles in multiple phases of axonal development. We recently identified afadin, an F-actin-binding protein, as an effector of R-Ras mediating axon branching through F-actin reorganization. Afadin comprises two isoforms--l-afadin, having the F-actin-binding domain, and s-afadin, lacking the F-actin-binding domain. Compared with l-afadin, s-afadin, the short splicing variant of l-afadin, contains RA domains but lacks the F-actin-binding domain. Neurons express both isoforms; however, the function of s-afadin in brain remains unknown. Here we identify s-afadin as an endogenous inhibitor of cortical axon branching. In contrast to the abundant and constant expression of l-afadin throughout neuronal development, the expression of s-afadin is relatively low when cortical axons branch actively. Ectopic expression and knockdown of s-afadin suppress and promote branching, respectively. s-Afadin blocks the R-Ras-mediated membrane translocation of l-afadin and axon branching by inhibiting the binding of l-afadin to R-Ras. Thus s-afadin acts as a dominant-negative isoform in R-Ras-afadin-regulated axon branching. PMID:25808489

  10. 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

  11. 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.

  12. 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

  13. 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

  14. 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.

  15. 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.

  16. 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

  17. 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

  18. 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

  19. 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 ...

  20. 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 ...

  1. 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 ...

  2. β-Actin-binding Complementarity-determining Region 2 of Variable Heavy Chain from Monoclonal Antibody C7 Induces Apoptosis in Several Human Tumor Cells and Is Protective against Metastatic Melanoma*

    PubMed Central

    Arruda, Denise C.; Santos, Luana C. P.; Melo, Filipe M.; Pereira, Felipe V.; Figueiredo, Carlos R.; Matsuo, Alisson L.; Mortara, Renato A.; Juliano, Maria A.; Rodrigues, Elaine G.; Dobroff, Andrey S.; Polonelli, Luciano; Travassos, Luiz R.

    2012-01-01

    Complementarity-determining regions (CDRs) from monoclonal antibodies tested as synthetic peptides display anti-infective and antitumor activities, independent of the specificity of the native antibody. Previously, we have shown that the synthetic peptide C7H2, based on the heavy chain CDR 2 from monoclonal antibody C7, a mAb directed to a mannoprotein of Candida albicans, significantly reduced B16F10 melanoma growth and lung colony formation by triggering tumor apoptosis. The mechanism, however, by which C7H2 induced apoptosis in tumor cells remained unknown. Here, we demonstrate that C7H2 interacts with components of the tumor cells cytoskeleton, being rapidly internalized after binding to the tumor cell surface. Mass spectrometry analysis and in vitro validation revealed that β-actin is the receptor of C7H2 in the tumor cells. C7H2 induces β-actin polymerization and F-actin stabilization, linked with abundant generation of superoxide anions and apoptosis. Major phenotypes following peptide binding were chromatin condensation, DNA fragmentation, annexin V binding, lamin disruption, caspase 8 and 3 activation, and organelle alterations. Finally, we evaluated the cytotoxic efficacy of C7H2 in a panel of human tumor cell lines. All tumor cell lines studied were equally susceptible to C7H2 in vitro. The C7H2 amide without further derivatization significantly reduced lung metastasis of mice endovenously challenged with B16F10-Nex2 melanoma cells. No significant cytotoxicity was observed toward nontumorigenic cell lines on short incubation in vitro or in naïve mice injected with a high dose of the peptide. We believe that C7H2 is a promising peptide to be developed as an anticancer drug. PMID:22334655

  3. 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

  4. 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

  5. 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

  6. 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

  7. 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

  8. 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.

  9. 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. ...

  10. 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 ...

  11. 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.

  12. 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

  13. 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...

  14. 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; ...

  15. [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

  16. 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.

  17. Soluble guanylyl cyclase-activated cyclic GMP-dependent protein kinase inhibits arterial smooth muscle cell migration independent of VASP-serine 239 phosphorylation.

    PubMed

    Holt, Andrew W; Martin, Danielle N; Shaver, Patti R; Adderley, Shaquria P; Stone, Joshua D; Joshi, Chintamani N; Francisco, Jake T; Lust, Robert M; Weidner, Douglas A; Shewchuk, Brian M; Tulis, David A

    2016-09-01

    Coronary artery disease (CAD) accounts for over half of all cardiovascular disease-related deaths. Uncontrolled arterial smooth muscle (ASM) cell migration is a major component of CAD pathogenesis and efforts aimed at attenuating its progression are clinically essential. Cyclic nucleotide signaling has long been studied for its growth-mitigating properties in the setting of CAD and other vascular disorders. Heme-containing soluble guanylyl cyclase (sGC) synthesizes cyclic guanosine monophosphate (cGMP) and maintains vascular homeostasis predominantly through cGMP-dependent protein kinase (PKG) signaling. Considering that reactive oxygen species (ROS) can interfere with appropriate sGC signaling by oxidizing the cyclase heme moiety and so are associated with several CVD pathologies, the current study was designed to test the hypothesis that heme-independent sGC activation by BAY 60-2770 (BAY60) maintains cGMP levels despite heme oxidation and inhibits ASM cell migration through phosphorylation of the PKG target and actin-binding vasodilator-stimulated phosphoprotein (VASP). First, using the heme oxidant ODQ, cGMP content was potentiated in the presence of BAY60. Using a rat model of arterial growth, BAY60 significantly reduced neointima formation and luminal narrowing compared to vehicle (VEH)-treated controls. In rat ASM cells BAY60 significantly attenuated cell migration, reduced G:F actin, and increased PKG activity and VASP Ser239 phosphorylation (pVASP·S239) compared to VEH controls. Site-directed mutagenesis was then used to generate overexpressing full-length wild type VASP (FL-VASP/WT), VASP Ser239 phosphorylation-mimetic (FL-VASP/239D) and VASP Ser239 phosphorylation-resistant (FL-VASP/239A) ASM cell mutants. Surprisingly, FL-VASP/239D negated the inhibitory effects of FL-VASP/WT and FL-VASP/239A cells on migration. Furthermore, when FL-VASP mutants were treated with BAY60, only the FL-VASP/239D group showed reduced migration compared to its VEH controls

  18. 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

  19. 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).

  20. 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

  1. 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

  2. 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

  3. 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 ...

  4. 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 ...

  5. 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: ...

  6. [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

  7. 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 ...

  8. 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.

  9. 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

  10. 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

  11. 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

  12. 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 ...

  13. 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…

  14. 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

  15. 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 ...

  16. 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 = ...

  17. 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…

  18. 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...

  19. 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.

  20. Sleep deprivation causes memory deficits by negatively impacting neuronal connectivity in hippocampal area CA1.

    PubMed

    Havekes, Robbert; Park, Alan J; Tudor, Jennifer C; Luczak, Vincent G; Hansen, Rolf T; Ferri, Sarah L; Bruinenberg, Vibeke M; Poplawski, Shane G; Day, Jonathan P; Aton, Sara J; Radwańska, Kasia; Meerlo, Peter; Houslay, Miles D; Baillie, George S; Abel, Ted

    2016-01-01

    Brief periods of sleep loss have long-lasting consequences such as impaired memory consolidation. Structural changes in synaptic connectivity have been proposed as a substrate of memory storage. Here, we examine the impact of brief periods of sleep deprivation on dendritic structure. In mice, we find that five hours of sleep deprivation decreases dendritic spine numbers selectively in hippocampal area CA1 and increased activity of the filamentous actin severing protein cofilin. Recovery sleep normalizes these structural alterations. Suppression of cofilin function prevents spine loss, deficits in hippocampal synaptic plasticity, and impairments in long-term memory caused by sleep deprivation. The elevated cofilin activity is caused by cAMP-degrading phosphodiesterase-4A5 (PDE4A5), which hampers cAMP-PKA-LIMK signaling. Attenuating PDE4A5 function prevents changes in cAMP-PKA-LIMK-cofilin signaling and cognitive deficits associated with sleep deprivation. Our work demonstrates the necessity of an intact cAMP-PDE4-PKA-LIMK-cofilin activation-signaling pathway for sleep deprivation-induced memory disruption and reduction in hippocampal spine density. PMID:27549340

  1. Sleep deprivation causes memory deficits by negatively impacting neuronal connectivity in hippocampal area CA1

    PubMed Central

    Havekes, Robbert; Park, Alan J; Tudor, Jennifer C; Luczak, Vincent G; Hansen, Rolf T; Ferri, Sarah L; Bruinenberg, Vibeke M; Poplawski, Shane G; Day, Jonathan P; Aton, Sara J; Radwańska, Kasia; Meerlo, Peter; Houslay, Miles D; Baillie, George S; Abel, Ted

    2016-01-01

    Brief periods of sleep loss have long-lasting consequences such as impaired memory consolidation. Structural changes in synaptic connectivity have been proposed as a substrate of memory storage. Here, we examine the impact of brief periods of sleep deprivation on dendritic structure. In mice, we find that five hours of sleep deprivation decreases dendritic spine numbers selectively in hippocampal area CA1 and increased activity of the filamentous actin severing protein cofilin. Recovery sleep normalizes these structural alterations. Suppression of cofilin function prevents spine loss, deficits in hippocampal synaptic plasticity, and impairments in long-term memory caused by sleep deprivation. The elevated cofilin activity is caused by cAMP-degrading phosphodiesterase-4A5 (PDE4A5), which hampers cAMP-PKA-LIMK signaling. Attenuating PDE4A5 function prevents changes in cAMP-PKA-LIMK-cofilin signaling and cognitive deficits associated with sleep deprivation. Our work demonstrates the necessity of an intact cAMP-PDE4-PKA-LIMK-cofilin activation-signaling pathway for sleep deprivation-induced memory disruption and reduction in hippocampal spine density. DOI: http://dx.doi.org/10.7554/eLife.13424.001 PMID:27549340

  2. 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

  3. 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.

  4. 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.

  5. 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.

  6. 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.

  7. 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

  8. 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

  9. 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

  10. 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.

  11. 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.

  12. Direct binding of F actin to the cytoplasmic domain of the alpha 2 integrin chain in vitro

    NASA Technical Reports Server (NTRS)

    Kieffer, J. D.; Plopper, G.; Ingber, D. E.; Hartwig, J. H.; Kupper, T. S.

    1995-01-01

    The transmembrane integrins have been shown to interact with the cytoskeleton via noncovalent binding between cytoplasmic domains (CDs) of integrin beta chains and various actin binding proteins within the focal adhesion complex. Direct or indirect integrin alpha chain CD binding to the actin cytoskeleton has not been reported. We show here that actin, as an abundant constituent of focal adhesion complex proteins isolated from fibroblasts, binds strongly and specifically to alpha 2 CD, but not to alpha 1 CD peptide. Similar specific binding to alpha 2 CD peptide was seen for highly purified F actin, free of putative actin-binding proteins. The bound complex of actin and peptide was visualized directly by coprecipitation, and actin binding was abrogated by removal of a five amino acid sequence from the alpha 2 CD peptide. Our findings may explain the earlier observation that, while integrins alpha 2 beta 1 and alpha 1 beta 1 both bind to collagen, only alpha 2 beta 1 can mediate contraction of extracellular collagen matrices.

  13. 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.

  14. 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

  15. 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

  16. 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

  17. 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

  18. 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.

  19. 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

  20. 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

  1. 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

  2. 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

  3. 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

  4. 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

  5. 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

  6. 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)

  7. 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

  8. 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

  9. 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.

  10. 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.

  11. 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

  12. 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.

  13. 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.

  14. 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

  15. 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

  16. 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.

  17. 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

  18. 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

  19. 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

  20. 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

  1. 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

  2. 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

  3. 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

  4. 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

  5. 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

  6. 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

  7. 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

  8. 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

  9. 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...

  10. 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.

  11. 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

  12. 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

  13. 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

  14. 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

  15. 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

  16. 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.

  17. 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

  18. 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

  19. 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

  20. 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.

  1. 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

  2. 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

  3. 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

  4. 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

  5. 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

  6. 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.

  7. (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.

  8. 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)

  9. 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.

  10. 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.

  11. 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

  12. 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

  13. 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.

  14. 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.

  15. 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.

  16. 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

  17. 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.

  18. Characterisation of Schizosaccharomyces pombe α-actinin

    PubMed Central

    Addario, Barbara; Sandblad, Linda; Persson, Karina

    2016-01-01

    The actin cytoskeleton plays a fundamental role in eukaryotic cells. Its reorganization is regulated by a plethora of actin-modulating proteins, such as a-actinin. In higher organisms, α-actinin is characterized by the presence of three distinct structural domains: an N-terminal actin-binding domain and a C-terminal region with EF-hand motif separated by a central rod domain with four spectrin repeats. Sequence analysis has revealed that the central rod domain of α-actinin from the fission yeast Schizosaccharomyces pombe consists of only two spectrin repeats. To obtain a firmer understanding of the structure and function of this unconventional α-actinin, we have cloned and characterized each structural domain. Our results show that this a-actinin isoform is capable of forming dimers and that the rod domain is required for this. However, its actin-binding and cross-linking activity appears less efficient compared to conventional α-actinins. The solved crystal structure of the actin-binding domain indicates that the closed state is stabilised by hydrogen bonds and a salt bridge not present in other α-actinins, which may reduce the affinity for actin. PMID:27069798

  19. Vinculin controls talin engagement with the actomyosin machinery

    PubMed Central

    Atherton, Paul; Stutchbury, Ben; Wang, De-Yao; Jethwa, Devina; Tsang, Ricky; Meiler-Rodriguez, Eugenia; Wang, Pengbo; Bate, Neil; Zent, Roy; Barsukov, Igor L.; Goult, Benjamin T.; Critchley, David R.; Ballestrem, Christoph

    2015-01-01

    The link between extracellular-matrix-bound integrins and intracellular F-actin is essential for cell spreading and migration. Here, we demonstrate how the actin-binding proteins talin and vinculin cooperate to provide this link. By expressing structure-based talin mutants in talin null cells, we show that while the C-terminal actin-binding site (ABS3) in talin is required for adhesion complex assembly, the central ABS2 is essential for focal adhesion (FA) maturation. Thus, although ABS2 mutants support cell spreading, the cells lack FAs, fail to polarize and exert reduced force on the surrounding matrix. ABS2 is inhibited by the preceding mechanosensitive vinculin-binding R3 domain, and deletion of R2R3 or expression of constitutively active vinculin generates stable force-independent FAs, although cell polarity is compromised. Our data suggest a model whereby force acting on integrin-talin complexes via ABS3 promotes R3 unfolding and vinculin binding, activating ABS2 and locking talin into an actin-binding configuration that stabilizes FAs. PMID:26634421

  20. Vinculin controls talin engagement with the actomyosin machinery.

    PubMed

    Atherton, Paul; Stutchbury, Ben; Wang, De-Yao; Jethwa, Devina; Tsang, Ricky; Meiler-Rodriguez, Eugenia; Wang, Pengbo; Bate, Neil; Zent, Roy; Barsukov, Igor L; Goult, Benjamin T; Critchley, David R; Ballestrem, Christoph

    2015-01-01

    The link between extracellular-matrix-bound integrins and intracellular F-actin is essential for cell spreading and migration. Here, we demonstrate how the actin-binding proteins talin and vinculin cooperate to provide this link. By expressing structure-based talin mutants in talin null cells, we show that while the C-terminal actin-binding site (ABS3) in talin is required for adhesion complex assembly, the central ABS2 is essential for focal adhesion (FA) maturation. Thus, although ABS2 mutants support cell spreading, the cells lack FAs, fail to polarize and exert reduced force on the surrounding matrix. ABS2 is inhibited by the preceding mechanosensitive vinculin-binding R3 domain, and deletion of R2R3 or expression of constitutively active vinculin generates stable force-independent FAs, although cell polarity is compromised. Our data suggest a model whereby force acting on integrin-talin complexes via ABS3 promotes R3 unfolding and vinculin binding, activating ABS2 and locking talin into an actin-binding configuration that stabilizes FAs. PMID:26634421

  1. HDAC6 Modulates Cell Motility by Altering the Acetylation Level of Cortactin

    PubMed Central

    Zhang, Xiaohong; Yuan, Zhigang; Zhang, Yingtao; Yong, Sarah; Salas-Burgos, Alexis; Koomen, John; Olashaw, Nancy; Parsons, J. Thomas; Yang, Xiang-Jiao; Dent, Sharon R.; Yao, Tso-Pang; Lane, William S.; Seto, Edward

    2009-01-01

    Summary Histone deacetylase 6 (HDAC6) is a tubulin-specific deacetylase that regulates microtubule-dependent cell movement. In this study, we identify the F-actin-binding protein, cortactin, as a HDAC6 substrate. We demonstrate that HDAC6 binds cortactin and that overexpression of HDAC6 leads to hypoacetylation of cortactin, while inhibition of HDAC6 activity leads to cortactin hyperacetylation. HDAC6 alters the ability of cortactin to bind F-actin by modulating a “charge patch” in its repeat region. Introduction of charge-preserving or charge-neutralizing mutations in this cortactin repeat region correlates with the gain or loss of F-actin binding ability, respectively. Cells expressing a charge-neutralizing cortactin mutant were less motile than control cells or cells expressing a charge-preserving mutant. These findings suggest that, in addition to its role in microtubule-dependent cell motility, HDAC6 influences actin-dependent cell motility by altering the acetylation status of cortactin, which, in turn, changes the F-actin binding activity of cortactin. PMID:17643370

  2. 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.

  3. 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.

  4. 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.

  5. 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.

  6. 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.

  7. 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

  8. 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 o