scholarly journals Purification and properties of transgelin: a transformation and shape change sensitive actin-gelling protein.

1993 ◽  
Vol 121 (5) ◽  
pp. 1065-1073 ◽  
Author(s):  
C Shapland ◽  
J J Hsuan ◽  
N F Totty ◽  
D Lawson
Keyword(s):  
Ionic Strength ◽  
Actin Filaments ◽  
Random Distribution ◽  
Shape Change ◽  
Protein A ◽  
Amino Acid Residues ◽  
Cell Permeabilization ◽  
Purification And Properties ◽  
Evolutionarily Conserved ◽  
Positively Charged

We have purified the transformation and shape change sensitive isoform of an actin associated polypeptide doublet previously described by us (Shapland, C., P. Lowings, and D. Lawson. 1988. J. Cell Biol. 107:153-161) and have shown that it is evolutionarily conserved as far back as yeast. The purified protein: (a) binds directly to actin filaments at a ratio of 1:6 actin monomers, with a binding constant (Ka) of approximately 7.5 x 10(5) M-1; and (b) causes actin filament gelation within 2 min. Although these activities are controlled by ionic strength (and may be mediated by positively charged amino acid residues) the molecule remains as a monomer irrespective of ionic conditions. EM reveals that the addition of this protein to actin filaments converts them from a loose, random distribution into a tangled, cross-linked meshwork within 1 min, and discrete tightly aggregated foci after 10 min. By use of an "add-back" cell permeabilization system we can rebind this molecule specifically to actin filaments in cells from which it has previously been removed. Since the protein is transformation sensitive and gels actin, we have named it transgelin.

Influence of nascent polypeptide positive charges on translation dynamics

2020 ◽  
Vol 477 (15) ◽  
pp. 2921-2934
Author(s):  
Rodrigo D. Requião ◽  
Géssica C. Barros ◽  
Tatiana Domitrovic ◽  
Fernando L. Palhano
Keyword(s):  
Mrna Decay ◽  
Translation Termination ◽  
Published Data ◽  
Translation Efficiency ◽  
Amino Acid Residues ◽  
High Concentration ◽  
Strong Argument ◽  
Translation Arrest ◽  
Exit Tunnel ◽  
Positively Charged

Protein segments with a high concentration of positively charged amino acid residues are often used in reporter constructs designed to activate ribosomal mRNA/protein decay pathways, such as those involving nonstop mRNA decay (NSD), no-go mRNA decay (NGD) and the ribosome quality control (RQC) complex. It has been proposed that the electrostatic interaction of the positively charged nascent peptide with the negatively charged ribosomal exit tunnel leads to translation arrest. When stalled long enough, the translation process is terminated with the degradation of the transcript and an incomplete protein. Although early experiments made a strong argument for this mechanism, other features associated with positively charged reporters, such as codon bias and mRNA and protein structure, have emerged as potent inducers of ribosome stalling. We carefully reviewed the published data on the protein and mRNA expression of artificial constructs with diverse compositions as assessed in different organisms. We concluded that, although polybasic sequences generally lead to lower translation efficiency, it appears that an aggravating factor, such as a nonoptimal codon composition, is necessary to cause translation termination events.

Binding of Human Platelet Glycoprotein lb and Actin to Fragments of Actin-Binding Protein

1992 ◽  
Vol 67 (02) ◽  
pp. 252-257 ◽  
Author(s):  
Anne M Aakhus ◽  
J Michael Wilkinson ◽  
Nils Olav Solum
Keyword(s):  
Actin Filaments ◽  
High Speed ◽  
Binding Protein ◽  
Protein A ◽  
Actin Binding ◽  
Platelet Glycoprotein ◽  
Actin Binding Protein ◽  
Crossed Immunoelectrophoresis ◽  
Triton X ◽  
Calpain Inhibitors

SummaryActin-binding protein (ABP) is degraded into fragments of 190 and 90 kDa by calpain. A monoclonal antibody (MAb TI10) against the 90 kDa fragment of ABP coprecipitated with the glycoprotein lb (GP lb) peak observed on crossed immunoelectrophoresis of Triton X-100 extracts of platelets prepared without calpain inhibitors. MAb PM6/317 against the 190 kDa fragment was not coprecipitated with the GP lb peak under such conditions. The 90 kDa fragment was adsorbed on protein A agarose from extracts that had been preincubated with antibodies to GP lb. This supports the idea that the GP Ib-ABP interaction resides in the 90 kDa region of ABP. GP lb was sedimented with the Triton-insoluble actin filaments in trace amounts only, and only after high speed centrifugation (100,000 × g, 3 h). Both the 190 kDa and the 90 kDa fragments of ABP were sedimented with the Triton-insoluble actin filaments.

scholarly journals Species-Specific Regulation of TRPM2 by PI(4,5)P2 via the Membrane Interfacial Cavity

2021 ◽  
Vol 22 (9) ◽  
pp. 4637
Author(s):  
Daniel Barth ◽  
Andreas Lückhoff ◽  
Frank J. P. Kühn
Keyword(s):  
Amino Acid Residues ◽  
Hek 293 ◽  
Complete Inactivation ◽  
293 Cells ◽  
Activation Mechanisms ◽  
Specific Regulation ◽  
Species Specific ◽  
Inside Out ◽  
Positively Charged

The human apoptosis channel TRPM2 is stimulated by intracellular ADR-ribose and calcium. Recent studies show pronounced species-specific activation mechanisms. Our aim was to analyse the functional effect of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), commonly referred to as PIP2, on different TRPM2 orthologues. Moreover, we wished to identify the interaction site between TRPM2 and PIP2. We demonstrate a crucial role of PIP2, in the activation of TRPM2 orthologues of man, zebrafish, and sea anemone. Utilizing inside-out patch clamp recordings of HEK-293 cells transfected with TRPM2, differential effects of PIP2 that were dependent on the species variant became apparent. While depletion of PIP2 via polylysine uniformly caused complete inactivation of TRPM2, restoration of channel activity by artificial PIP2 differed widely. Human TRPM2 was the least sensitive species variant, making it the most susceptible one for regulation by changes in intramembranous PIP2 content. Furthermore, mutations of highly conserved positively charged amino acid residues in the membrane interfacial cavity reduced the PIP2 sensitivity in all three TRPM2 orthologues to varying degrees. We conclude that the membrane interfacial cavity acts as a uniform PIP2 binding site of TRPM2, facilitating channel activation in the presence of ADPR and Ca2+ in a species-specific manner.

Block of Human Heart hH1 Sodium Channels by the Enantiomers of Bupivacaine

2000 ◽  
Vol 93 (4) ◽  
pp. 1022-1033 ◽  
Author(s):  
Carla Nau ◽  
Sho-Ya Wang ◽  
Gary R. Strichartz ◽  
Ging Kuo Wang
Keyword(s):  
Human Heart ◽  
Point Mutations ◽  
Cell Voltage ◽  
Site Directed Mutagenesis ◽  
Na Channel ◽  
Amino Acid Residues ◽  
Na Channels ◽  
State Dependent ◽  
Positively Charged

Background S(-)-bupivacaine reportedly exhibits lower cardiotoxicity but similar local anesthetic potency compared with R(+)-bupivacaine. The bupivacaine binding site in human heart (hH1) Na+ channels has not been studied to date. The authors investigated the interaction of bupivacaine enantiomers with hH1 Na+ channels, assessed the contribution of putatively relevant residues to binding, and compared the intrinsic affinities to another isoform, the rat skeletal muscle (mu1) Na+ channel. Methods Human heart and mu1 Na+ channel alpha subunits were transiently expressed in HEK293t cells and investigated during whole cell voltage-clamp conditions. Using site-directed mutagenesis, the authors created point mutations at positions hH1-F1760, hH1-N1765, hH1-Y1767, and hH1-N406 by introducing the positively charged lysine (K) or the negatively charged aspartic acid (D) and studied their influence on state-dependent block by bupivacaine enantiomers. Results Inactivated hH1 Na+ channels displayed a weak stereoselectivity with a stereopotency ratio (+/-) of 1.5. In mutations hH1-F1760K and hH1-N1765K, bupivacaine affinity of inactivated channels was reduced by approximately 20- to 40-fold, in mutation hH1-N406K by approximately sevenfold, and in mutations hH1-Y1767K and hH1-Y1767D by approximately twofold to threefold. Changes in recovery of inactivated mutant channels from block paralleled those of inactivated channel affinity. Inactivated hH1 Na+ channels exhibited a slightly higher intrinsic affinity than mu1 Na+ channels. Conclusions Differences in bupivacaine stereoselectivity and intrinsic affinity between hH1 and mu1 Na+ channels are small and most likely of minor clinical relevance. Amino acid residues in positions hH1-F1760, hH1-N1765, and hH1-N406 may contribute to binding of bupivacaine enantiomers in hH1 Na+ channels, whereas the role of hH1-Y1767 remains unclear.

scholarly journals Positively charged amino acid residues located similarly in sea anemone and scorpion toxins

1994 ◽  
Vol 269 (24) ◽  
pp. 16785-16788
Author(s):  
E.P. Loret ◽  
R.M. del Valle ◽  
P. Mansuelle ◽  
F. Sampieri ◽  
H. Rochat
Keyword(s):  
Amino Acid ◽  
Sea Anemone ◽  
Amino Acid Residues ◽  
Scorpion Toxins ◽  
Positively Charged

scholarly journals Sequence and Structure of Human Rhinoviruses Reveal the Basis of Receptor Discrimination

2003 ◽  
Vol 77 (12) ◽  
pp. 6923-6930 ◽  
Author(s):  
Marketa Vlasak ◽  
Soile Blomqvist ◽  
Tapani Hovi ◽  
Elizabeth Hewat ◽  
Dieter Blaas
Keyword(s):  
Low Density Lipoprotein ◽  
Three Dimensional ◽  
Density Lipoprotein ◽  
Major Group ◽  
Amino Acid Residues ◽  
X Ray ◽  
Vldl Receptor ◽  
Capsid Protein Vp1 ◽  
Three Dimensional Models ◽  
Positively Charged

ABSTRACT The sequences of the capsid protein VP1 of all minor receptor group human rhinoviruses were determined. A phylogenetic analysis revealed that minor group HRVs were not more related to each other than to the nine major group HRVs whose sequences are known. Examination of the surface exposed amino acid residues of HRV1A and HRV2, whose X-ray structures are available, and that of three-dimensional models computed for the remaining eight minor group HRVs indicated a pattern of positively charged residues within the region, which, in HRV2, was shown to be the binding site of the very-low-density lipoprotein (VLDL) receptor. A lysine in the HI loop of VP1 (K224 in HRV2) is strictly conserved within the minor group. It lies in the middle of the footprint of a single repeat of the VLDL receptor on HRV2. Major group virus serotypes exhibit mostly negative charges at the corresponding positions and do not bind the negatively charged VLDL receptor, presumably because of charge repulsion.

scholarly journals Rules for the self-assembly of ESCRT-III on endosomes

2021 ◽  
Author(s):  
Simon Sprenger ◽  
Simona M. Migliano ◽  
Florian Oleschko ◽  
Marvin Kobald ◽  
Michael Hess ◽  
...  
Keyword(s):  
Self Assembly ◽  
Hydrophobic Interactions ◽  
The Self ◽  
Amino Acid Residues ◽  
Lateral Expansion ◽  
Endosomal Sorting ◽  
Aaa Atpase ◽  
Charged Amino Acids ◽  
Membrane Budding ◽  
Positively Charged

ABSTRACTThe endosomal sorting complexes required for transport (ESCRT) mediate various membrane remodeling processes in cells by mechanism that are incompletely understood. Here we combined genetic experiments in budding yeast with site-specific cross-linking to identify rules that govern the self-assembly of individual ESCRT-III proteins into functional ESCRT-III complexes on endosomes. Together with current structural models of ESCRT-III, our findings suggest that, once nucleated, the growing Snf7 protofilament seeds the lateral co-assembly of a Vps24 - Vps2 heterofilament. Both Vps24 and Vps2 use positively charged amino acid residues in their helices α1 to interact with negatively charged amino acids in helix α4 of Snf7 subunits of the protofilament. In the Vps24 - Vps2 heterofilament, the two subunits alternate and interact with each other using hydrophobic interactions between helices α2/α3. The co-assembly of the Vps24 - Vps2 heterofilament restricts the lateral expansion of Snf7 protofilaments and leads the immediate recruitment of the AAA-ATPase Vps4. This self-assembly process of three ESCRT-III subunits results in the formation of a Snf7 protofilament and the co-assembly of a Vps24 - Vps2 heterofilament. This sets the stage for Vps4 recruitment and the subsequent ATP-driven dynamic self-organization of ESCRT-III / Vps4 assemblies and the ensuing membrane budding and scission events.

scholarly journals Effects of external pH on substrate binding and on the inward chloride translocation rate constant of band 3.

1996 ◽  
Vol 107 (2) ◽  
pp. 271-291 ◽  
Author(s):  
S Q Liu ◽  
F Y Law ◽  
P A Knauf
Keyword(s):  
Rate Constant ◽  
Band 3 ◽  
Complex Model ◽  
Amino Acid Residues ◽  
Ping Pong ◽  
Extracellular Transport ◽  
Charged Form ◽  
Transport Site ◽  
External Ph ◽  
Positively Charged

To test the hypothesis that amino acid residues in band 3 with titratable positive charges play a role in the binding of anions to the outside-facing transport site, we measured the effects of changing external pH (pH(O)) on the dissociation constant for binding of external iodide to the transport site, K(O)(I). K(O)(I) increased with increasing pH(O), and a significant increase was seen even at pH(O) values as low as 9.9. The dependence of K(O)(I) on pH(O) can be explained by a model with one titratable site with pK 9.5 +/- 0.2 (probably lysine), which increases anion affinity for the external transport site when it is in the positively charged form. A more complex model, analogous to one recently proposed by Bjerrum (1992), with two titratable sites, one with pK 9.3 +/- 0.3 (probably lysine) and another with pK > 11 (probably arginine), gives a slightly better fit to the data. Thus, titratable positively charged residues seem to be functionally important for the binding of substrate anions to the outward-facing anion transport site. In addition, analysis of Dixon plot slopes for L inhibition of Cl- exchange at different pH 0 values, coupled with the assumption that pH(O) has parallel effects on external I- and Cl- binding, indicates that k', the rate-constant for inward translocation of the complex of Cl- with the extracellular transport site, decreases with increasing pH(O). The data are compatible with a model in which titration of the pK 9.3 residue decreases k to 14 +/- 10% of its value at neutral pH(O). This result, however, together with Bjerrum's (1992) observation that the maximum flux J(M)) increases 1.6-fold when this residue is deprotonated, makes quantitative predictions that raise significant questions about the adequacy of the two titratable site ping-pong model or the assumptions used in analyzing the data.

Fibrinogen C-terminal peptidic sequences (Haptides) modulate fibrin polymerization

2004 ◽  
Vol 91 (01) ◽  
pp. 43-51 ◽  
Author(s):  
Matti Ben-Moshe ◽  
Sholomo Magdassi ◽  
Raphael Gorodetsky ◽  
Gerard Marx
Keyword(s):  
Platelet Aggregation ◽  
Cell Attachment ◽  
Fibrin Clot ◽  
Nano Particles ◽  
Amino Acid Residues ◽  
Fibrin Gel ◽  
Transmission Microscopy ◽  
Average Size ◽  
Β Chain ◽  
Positively Charged

SummaryWe previously described synthetic peptides of 19-21 amino acid residues, homologous to the C-termini of fibrinogen Fib340 and Fib420, from the β-chain (Cβ), the extended αE chain (CαE) and near the end of the γ-chain (preCγ) which elicited attachment (haptotactic) responses from mesenchymal cells. We named these haptotactic peptides -Haptides. The effects of Haptides on fibrin clot formation was evaluated and their possible effects on platelet aggregation was examined. The Haptides Cβ, CαE and preCγ, (2-10 μM) increased fibrin clot turbidity and also decreased thrombin-induced clotting time. Higher concentrations (>120 μM of Cβ or preCγ) induced fibrinogen precipitation even without thrombin. These precipitates exhibited different ultrastructure from thrombin-induced fibrin by scanning and transmission microscopy. C-terminal peptides of the other fibrinogen chains exerted no such effects. Sepharose beads covalently coated with either whole fibrinogen or Haptides (SB-Fib or SB-Haptide) highly adsorbed free FITCHaptides. In aqueous solution, Haptides formed nano-particles with average size of ∼150nm in diameter. We suggest that such positively charged aggregates could serve to nucleate and accelerate fibrin gel formation. These results also indicate that Cβ and preCγ sequences within fibrin(ogen) participate in the docking and condensation of fibrin(ogen) during its assembly into a fibrin clot. By contrast, Haptides up to 100µM did not bind to platelets, and had no effect on platelet aggregation. Our findings highlight the roles of the C-terminal sequences of the β and γ chains in fibrin(ogen) polymerization as well as in cell attachment.

Sign in / Sign up

Export Citation Format

Share Document