Coronin localizes to leading edges and is involved in cell spreading and lamellipodium extension in vertebrate cells

1999 ◽  
Vol 112 (17) ◽  
pp. 2833-2842 ◽  
Author(s):  
M. Mishima ◽  
E. Nishida
Keyword(s):  
Amino Acids ◽  
Cell Spreading ◽  
Immunofluorescent Staining ◽  
Actin Binding ◽  
Cellular Slime Mold ◽  
Cell Periphery ◽  
Terminal Amino ◽  
Wd Repeat ◽  
Leading Edges

Coronin is a WD repeat-containing actin-binding protein, which was originally identified in the cellular slime mold Dictyostelium. Coronin-null Dictyostelium cells show defects in cytokinesis, cell motility and phagocytosis. Although the existence of coronin in higher eukaryotes has been reported, its function in vertebrate cells has not been elucidated. We cloned a Xenopus homolog of coronin (Xcoronin) and examined its actin-binding properties, subcellular localization and possible functions. Xcoronin consists of 480 amino acids and is 63% identical to human coronin (p57). Bacterially expressed recombinant Xcoronin co-sedimented with F-actin in vitro. The WD repeat domain (residues 64–299) alone did not have any affinity for F-actin. Anti-Xcoronin antibodies reacted specifically with a single 57 kDa protein present in an extract of the Xenopus A6 cell line. Indirect immunofluorescent staining of A6 cells revealed that Xcoronin is present in the cytoplasm and concentrated in the cell periphery in membrane ruffles. During spreading after replating or wound healing after scratching a confluent monolayer, Xcoronin became concentrated in the leading edges of lamellipodia. A GFP-fusion protein of Xcoronin showed a subcellular distribution essentially identical to endogenous Xcoronin. The localization of Xcoronin to the cell periphery was resistant to treatment with 0.1% Triton X-100. The deletion of 63 N-terminal amino acids or of 65 C-terminal amino acids abolished the localization of Xcoronin to the cell periphery. Xcoronin expressed in 3T3 fibroblasts was concentrated to the leading edges of lamellipodia induced by active Rac. Remarkably, expression of a truncated form of Xcoronin (64–299), but not of full-length Xcoronin, significantly decreased the rate of cell spreading after replating and markedly inhibited lamellipodium extension induced by active Rac. These results suggest that Xcoronin plays an important role in lamellipodium extension and cell spreading.

scholarly journals Modular structure of chromosomal proteins HMG-14 and HMG-17: definition of a transcriptional enhancement domain distinct from the nucleosomal binding domain.

1995 ◽  
Vol 15 (12) ◽  
pp. 6663-6669 ◽  
Author(s):  
L Trieschmann ◽  
Y V Postnikov ◽  
A Rickers ◽  
M Bustin
Keyword(s):  
Amino Acids ◽  
Structural Features ◽  
Binding Domain ◽  
Full Length ◽  
Modular Structure ◽  
Chromosomal Proteins ◽  
Nucleosome Core ◽  
Terminal Amino

Chromosomal proteins HMG-14 and HMG-17 are the only known nuclear proteins which specifically bind to the nucleosome core particle and are implicated in the generation and/or maintenance of structural features specific to active chromatin. The two proteins facilitate polymerase II and III transcription from in vitro- and in vivo-assembled circular chromatin templates. Here we used deletion mutants and specific peptides to identify the transcriptional enhancement domain and delineate the nucleosomal binding domain of the HMG-14 and -17 proteins. Deletion of the 22 C-terminal amino acids of HMG-17 or 26 C-terminal amino acids of HMG-14 reduces significantly the ability of the proteins to enhance transcription from chromatin templates. In contrast, N-terminal truncation mutants had the same transcriptional enhancement activity as the full-length proteins. We conclude that the negatively charged C-terminal region of the proteins is required for transcriptional enhancement. Chromatin transcription enhancement assays, which involve binding competition between the full-length proteins and peptides derived from their nucleosomal binding regions, indicate that the minimal nucleosomal binding domain of human HMG-17 is 24 amino acids long and spans residues 17 to 40. The results suggest that HMG-14 and -17 proteins have a modular structure and contain distinct functional domains.

scholarly journals Hepatitis C Virus Nonstructural 5A Protein Induces Interleukin-8, Leading to Partial Inhibition of the Interferon-Induced Antiviral Response

2001 ◽  
Vol 75 (13) ◽  
pp. 6095-6106 ◽  
Author(s):  
Stephen J. Polyak ◽  
Khalid S. A. Khabar ◽  
Denise M. Paschal ◽  
Heather J. Ezelle ◽  
Gilles Duverlie ◽  
...  
Keyword(s):  
Amino Acids ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Antiviral Response ◽  
Full Length ◽  
Interleukin 8 ◽  
Antiviral Resistance ◽  
Ns5a Protein ◽  
Terminal Amino

ABSTRACT Hepatitis C virus (HCV), a major cause of liver disease worldwide, is frequently resistant to the antiviral alpha interferon (IFN). The HCV nonstructural 5A (NS5A) protein has been implicated in HCV antiviral resistance in many studies. NS5A antagonizes the IFN antiviral response in vitro, and one mechanism is via inhibition of a key IFN-induced enzyme, the double-stranded-RNA-activated protein kinase (PKR). In the present study we determined if NS5A uses other strategies to subvert the IFN system. Expression of full-length NS5A proteins from patients who exhibited a complete response (FL-NS5A-CR) or were nonresponsive (FL-NS5A-NR) to IFN therapy in HeLa cells had no effect on IFN induction of IFN-stimulated gene factor 3 (ISGF-3). Expression of mutant NS5A proteins lacking 110 (NS5A-ΔN110), 222 (NS5A-ΔN222), and 334 amino-terminal amino acids and mutants lacking 117 and 230 carboxy-terminal amino acids also had no effect on ISGF-3 induction by IFN. Expression of FL-NS5A-CR and FL-NS5A-NR did not affect IFN-induced STAT-1 tyrosine phosphorylation or upregulation of PKR and major histocompatibility complex class I antigens. However, NS5A expression in human cells induced interleukin 8 (IL-8) mRNA and protein, and this effect correlated with inhibition of the antiviral effects of IFN in an in vitro bioassay. NS5A induced transcription of a reporter gene driven by the IL-8 promoter, and the first 133 bp of the IL-8 promoter made up the minimal domain required for NS5A transactivation. NS5A-ΔN110 and NS5A-ΔN222 stimulated the IL-8 promoter to higher levels than did the full-length NS5A protein, and this correlated with increased nuclear localization of the proteins. Additional mutagenesis of the IL-8 promoter suggested that NF-κB and AP-1 were important in NS5A-ΔN222 transactivation in the presence of tumor necrosis factor alpha and that NF–IL-6 was inhibitory to this process. This study suggests that NS5A inhibits the antiviral actions of IFN by at least two mechanisms and provides the first evidence for a biological effect of the transcriptional activity of the NS5A protein. During HCV infection, viral proteins may induce chemokines that contribute to HCV antiviral resistance and pathogenesis.

scholarly journals The lymphocyte-specific protein LSP1 binds to F-actin and to the cytoskeleton through its COOH-terminal basic domain.

1992 ◽  
Vol 118 (6) ◽  
pp. 1443-1453 ◽  
Author(s):  
J Jongstra-Bilen ◽  
P A Janmey ◽  
J H Hartwig ◽  
S Galea ◽  
J Jongstra
Keyword(s):  
Amino Acids ◽  
Cell Line ◽  
Fusion Proteins ◽  
Specific Protein ◽  
Lymphoma Cell ◽  
Actin Binding ◽  
Lymphoma Cell Line ◽  
Basic Domain ◽  
Acidic Domain

The lymphocyte-specific phosphoprotein LSP1 associates with the cytoplasmic face of the plasma membrane and with the cytoskeleton. Mouse LSP1 protein contains 330 amino acids and contains an NH2-terminal acidic domain of approximately 177 amino acids. The COOH-terminal half of the LSP1 protein is rich in basic residues. In this paper we show that LSP1 protein which is immunoprecipitated with anti-LSP1 antibodies from NP-40-soluble lysates of the mouse B-lymphoma cell line BAL17 is associated with actin. In vitro binding experiments using recombinant LSP1 (rLSP1) protein and rabbit skeletal muscle actin show that LSP1 binds along the sides of F-actin but does not bind to G-actin. rLSP1 does not alter the initial polymerization kinetics of actin. The highly conserved COOH-terminal basic domains of mouse and human LSP1 share a significant homology with the 20-kD COOH-terminal F-actin binding fragment of caldesmon. A truncated rLSP1 protein containing the entire COOH-terminal basic domain from residue 179 to 330, but not the NH2-terminal acidic domain binds to F-actin at least as well as rLSP1. When LSP1/CAT fusion proteins are expressed in a LSP1-negative T-lymphoma cell line, only fusion proteins containing the basic COOH-terminal domain associate with the NP-40-insoluble cytoskeleton. These data show that LSP1 binds F-actin through its COOH-terminal basic domain and strongly suggest that LSP1 interacts with the cytoskeleton by direct binding to F-actin. We propose that LSP1 plays a role in mediating cytoskeleton driven responses in lymphocytes such as receptor capping, cell motility, or cell-cell interactions.

New Insights into the Function of N-Terminal 11 Amino Acids of Band 3 from Structural and Functional Study of a Naturally Occuring Band 3 Variant.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 577-577 ◽  
Author(s):  
Silverio Perrotta ◽  
Borriello Adriana ◽  
Lucia De Franceschi ◽  
Bruno Nobili ◽  
Achille Iolascon ◽  
...  
Keyword(s):  
Amino Acids ◽  
Band 3 ◽  
Red Cell ◽  
Red Cell Membrane ◽  
Exon 2 ◽  
Protein 4.2 ◽  
Membrane Bound ◽  
Terminal Amino ◽  
Intron 2

Abstract The 911 amino acid human erythroid AE1 (eAE1) Cl-/HCO3- exchanger SLC4A1 (usually called band 3) is the major intrinsic membrane protein of red cells. The N-terminal cytoplasmic domain of AE1 represents the anchoring site for membrane-associated proteins such as ankyrin, protein 4.2, protein 4.1, glycolytic enzymes (including aldolase and glyceraldeyde-3-phosphate dehydrogenase (GAPDH) and hemoglobin. We identified marked band 3 deficiency in the second son of a consanguineous marriage with a life-threatening nonimmune hemolytic anemia. The patient was transfusion-dependent prior to splenectomy. SDS-PAGE and immunoblotting analysis of the proband red cell membrane proteins showed approximately 12±4% of band 3 and protein 4.2 compared to controls. Direct nucleotide sequence of SLC4A1 gene showed a single base substitution (T->C) at position +2 in the donor splice site of intron 2 (Band 3 Neapolis). Functionally, the mutation causes an altered splicing with the consequent formation of two different mature mRNAs, one including intron 2 and one skipping exon 2. While intron 2 retention leads to premature translation termination, exon 2 skipping causes the loss of the normal start site of eAE1 protein translation. The purification of mutant band 3 and its characterization by MALDI mass spectrometry demonstrated the lack of the first 11 amino acids due to the usage of second in frame start site. Real-time RT-PCR analyses of reticulocyte mRNA showed a marked decrement in band 3 transcription accounting for protein deficiency. The lack of the 11 N-terminal amino acids resulted in complete absence of membrane bound aldolase while other glycolitic enzymes (for example GAPDH) were membrane bound. Syk tyrosine kinase recognized the truncated band 3 as a substrate in vitro. In spite of this ability to be phosphorylated by Syk and to recruit Lyn tyrosine kinase in vitro, we were unable to demonstrate Tyr-phosphorylation of mutant band 3 in intact erythrocytes following stimulation by oxidative stress. This finding implies a requirement for the 11 N-terminal amino acids for the sequential Tyr-phosphorylation of band 3 in intact red cell membranes. The mutant band 3 was largely present in the high molecular weight aggregate fraction (about 5.2 fold higher than control), indicating its increased tendency to cluster in the membrane. The spontaneous clustering of truncated band 3 strongly suggests that the negatively charged N-terminal domain may regulate oligomeric state of band 3 in the membrane. Biophysical characterization showed that band 3 deficiency resulted in decreased cohesion between lipid bilyer and spectrin based membrane skeleton accounting for membrane loss. The structural and functional characterization of the naturally occuring mutant band 3 has enabled us to identify a significant role for the 11 N-terminal amino acids in band 3 function and in red cell membrane physiology.

scholarly journals Physicochemical characterization of carbamylated human serum albumin: an in vitro study

RSC Advances ◽  
2019 ◽  
Vol 9 (63) ◽  
pp. 36508-36516
Author(s):  
Asim Badar ◽  
Zarina Arif ◽  
Shireen Naaz Islam ◽  
Khursheed Alam
Keyword(s):  
Amino Acids ◽  
Human Serum Albumin ◽  
Serum Albumin ◽  
Human Serum ◽  
Physicochemical Characterization ◽  
In Vitro Study ◽  
Terminal Amino ◽  
Carbamyl Amino Acids

Carbamylation is an ubiquitous process in which cyanate (OCN−) reacts with the N-terminal amino or ε-amino moiety and generates α-carbamyl amino acids and ε-carbamyl-lysine (homocitrulline).

scholarly journals Separate information required for nuclear and subnuclear localization: additional complexity in localizing an enzyme shared by mitochondria and nuclei.

1992 ◽  
Vol 12 (12) ◽  
pp. 5652-5658 ◽  
Author(s):  
A M Rose ◽  
P B Joyce ◽  
A K Hopper ◽  
N C Martin
Keyword(s):  
Amino Acids ◽  
Nuclear Periphery ◽  
Immunofluorescent Staining ◽  
Coding Region ◽  
Nuclear Targeting ◽  
Subnuclear Localization ◽  
Amino Terminal ◽  
Linear Sequence ◽  
Trna Methyltransferase ◽  
Terminal Amino

The TRM1 gene of Saccharomyces cerevisiae codes for a tRNA modification enzyme, N2,N2-dimethylguanosine-specific tRNA methyltransferase (m2(2)Gtase), shared by mitochondria and nuclei. Immunofluorescent staining at the nuclear periphery demonstrates that m2(2)Gtase localizes at or near the nuclear membrane. In determining sequences necessary for targeting the enzyme to nuclei and mitochondria, we found that information required to deliver the enzyme to the nucleus is not sufficient for its correct subnuclear localization. We also determined that mislocalizing the enzyme from the nucleus to the cytoplasm does not destroy its biological function. This change in location was caused by altering a sequence similar to other known nuclear targeting signals (KKSKKKRC), suggesting that shared enzymes are likely to use the same import pathway as proteins that localize only to the nucleus. As with other well-characterized mitochondrial proteins, the mitochondrial import of the shared methyltransferase depends on amino-terminal amino acids, and removal of the first 48 amino acids prevents its import into mitochondria. While this truncated protein is still imported into nuclei, the immunofluorescent staining is uniform throughout rather than at the nuclear periphery, a staining pattern identical to that described for a fusion protein consisting of the first 213 amino acids of m2(2)Gtase in frame with beta-galactosidase. As both of these proteins together contain the entire m2(2)Gtase coding region, the information necessary for association with the nuclear periphery must be more complex than the short linear sequence necessary for nuclear localization.

scholarly journals Mutational analysis of yeast profilin.

1993 ◽  
Vol 13 (12) ◽  
pp. 7864-7873 ◽  
Author(s):  
B K Haarer ◽  
A S Petzold ◽  
S S Brown
Keyword(s):  
Amino Acids ◽  
Adenylate Cyclase ◽  
Mutational Analysis ◽  
Actin Binding ◽  
In Vitro Assays ◽  
C Terminus ◽  
Physiological Relevance ◽  
In Vivo Effects

We have mutated two regions within the yeast profilin gene in an effort to functionally dissect the roles of actin and phosphatidylinositol 4,5-bisphosphate (PIP2) binding in profilin function. A series of truncations was carried out at the C terminus of profilin, a region that has been implicated in actin binding. Removal of the last three amino acids nearly eliminated the ability of profilin to bind polyproline in vitro but had no dramatic in vivo effects. Thus, the extreme C terminus is implicated in polyproline binding, but the physiological relevance of this interaction is called into question. More extensive truncation, of up to eight amino acids, had in vivo effects of increasing severity and resulted in changes in conformation and expression level of the mutant profilins. However, the ability of these mutants to bind actin in vitro was not eliminated, suggesting that this region cannot be solely responsible for actin binding. We also mutagenized a region of profilin that we hypothesized might be involved in PIP2 binding. Alteration of basic amino acids in this region produced mutant profilins that functioned well in vivo. Many of these mutants, however, were unable to suppress the loss of adenylate cyclase-associated protein (Cap/Srv2p [A. Vojtek, B. Haarer, J. Field, J. Gerst, T. D. Pollard, S. S. Brown, and M. Wigler, Cell 66:497-505, 1991]), indicating that a defect could be demonstrated in vivo. In vitro assays demonstrated that the inability to suppress loss of Cap/Srv2p correlated with a defect in the interaction with actin, independently of whether PIP2 binding was reduced. Since our earlier studies of Acanthamoeba profilins suggested the importance of PIP2 binding for suppression, we conclude that both activities are implicated and that an interplay between PIP2 binding and actin binding may be important for profilin function.

scholarly journals Loss of cargo binding in the human myosin VI deafness mutant (R1166X) leads to increased actin filament binding

2016 ◽  
Vol 473 (19) ◽  
pp. 3307-3319 ◽  
Author(s):  
Susan D. Arden ◽  
David A. Tumbarello ◽  
Tariq Butt ◽  
John Kendrick-Jones ◽  
Folma Buss
Keyword(s):  
Amino Acids ◽  
Adaptor Protein ◽  
Adaptor Proteins ◽  
Actin Binding ◽  
Binding Motif ◽  
Myosin Vi ◽  
Binding Ability ◽  
Activated State ◽  
Cargo Binding

Mutations in myosin VI have been associated with autosomal-recessive (DFNB37) and autosomal-dominant (DFNA22) deafness in humans. Here, we characterise an myosin VI nonsense mutation (R1166X) that was identified in a family with hereditary hearing loss in Pakistan. This mutation leads to the deletion of the C-terminal 120 amino acids of the myosin VI cargo-binding domain, which includes the WWY-binding motif for the adaptor proteins LMTK2, Tom1 as well as Dab2. Interestingly, compromising myosin VI vesicle-binding ability by expressing myosin VI with the R1166X mutation or with single point mutations in the adaptor-binding sites leads to increased F-actin binding of this myosin in vitro and in vivo. As our results highlight the importance of cargo attachment for regulating actin binding to the motor domain, we perform a detailed characterisation of adaptor protein binding and identify single amino acids within myosin VI required for binding to cargo adaptors. We not only show that the adaptor proteins can directly interact with the cargo-binding tail of myosin VI, but our in vitro studies also suggest that multiple adaptor proteins can bind simultaneously to non-overlapping sites in the myosin VI tail. In conclusion, our characterisation of the human myosin VI deafness mutant (R1166X) suggests that defects in cargo binding may leave myosin VI in a primed/activated state with an increased actin-binding ability.

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