scholarly journals Novel Interactions of ESCRT-III with LIP5 and VPS4 and their Implications for ESCRT-III Disassembly

2008 ◽  
Vol 19 (6) ◽  
pp. 2661-2672 ◽  
Author(s):  
Soomin Shim ◽  
Samuel A. Merrill ◽  
Phyllis I. Hanson
Keyword(s):  
Atpase Activity ◽  
Binding Site ◽  
Binding Sites ◽  
Essential Role ◽  
Multivesicular Body ◽  
The Other ◽  
Aaa Atpase ◽  
Direct Binding ◽  
Novel Interactions

The AAA+ ATPase VPS4 plays an essential role in multivesicular body biogenesis and is thought to act by disassembling ESCRT-III complexes. VPS4 oligomerization and ATPase activity are promoted by binding to LIP5. LIP5 also binds to the ESCRT-III like protein CHMP5/hVps60, but how this affects its function remains unclear. Here we confirm that LIP5 binds tightly to CHMP5, but also find that it binds well to additional ESCRT-III proteins including CHMP1B, CHMP2A/hVps2–1, and CHMP3/hVps24 but not CHMP4A/hSnf7–1 or CHMP6/hVps20. LIP5 binds to a different region within CHMP5 than within the other ESCRT-III proteins. In CHMP1B and CHMP2A, its binding site encompasses sequences at the proteins' extreme C-termini that overlap with “MIT interacting motifs” (MIMs) known to bind to VPS4. We find unexpected evidence of a second conserved binding site for VPS4 in CHMP2A and CHMP1B, suggesting that LIP5 and VPS4 may bind simultaneously to these proteins despite the overlap in their primary binding sites. Finally, LIP5 binds preferentially to soluble CHMP5 but instead to polymerized CHMP2A, suggesting that the newly defined interactions between LIP5 and ESCRT-III proteins may be regulated by ESCRT-III conformation. These studies point to a role for direct binding between LIP5 and ESCRT-III proteins that is likely to complement LIP5's previously described ability to regulate VPS4 activity.

E-box- and MEF-2-independent muscle-specific expression, positive autoregulation, and cross-activation of the chicken MyoD (CMD1) promoter reveal an indirect regulatory pathway

1994 ◽  
Vol 14 (8) ◽  
pp. 5474-5486
Author(s):  
C A Dechesne ◽  
Q Wei ◽  
J Eldridge ◽  
L Gannoun-Zaki ◽  
P Millasseau ◽  
...  
Keyword(s):  
Binding Site ◽  
Binding Sites ◽  
Primary Cultures ◽  
Regulatory Elements ◽  
The Other ◽  
Indirect Pathway ◽  
Specific Expression ◽  
Fibroblast Cultures ◽  
E Box ◽  
Myogenic Factors

Members of the MyoD family of gene-regulatory proteins (MyoD, myogenin, myf5, and MRF4) have all been shown not only to regulate the transcription of numerous muscle-specific genes but also to positively autoregulate and cross activate each other's transcription. In the case of muscle-specific genes, this transcriptional regulation can often be correlated with the presence of a DNA consensus in the regulatory region CANNTG, known as an E box. Little is known about the regulatory interactions of the myogenic factors themselves; however, these interactions are thought to be important for the activation and maintenance of the muscle phenotype. We have identified the minimal region in the chicken MyoD (CMD1) promoter necessary for muscle-specific transcription in primary cultures of embryonic chicken skeletal muscle. The CMD1 promoter is silent in primary chick fibroblast cultures and in muscle cell cultures treated with the thymidine analog bromodeoxyuridine. However, CMD1 and chicken myogenin, as well as, to a lesser degree, chicken Myf5 and MRF4, expressed in trans can activate transcription from the minimal CMD1 promoter in these primary fibroblast cultures. Here we show that the CMD1 promoter contains numerous E-box binding sites for CMD1 and the other myogenic factors, as well as a MEF-2 binding site. Surprisingly, neither muscle-specific and the other myogenic factors, as well as a MEF-2 binding site. Surprisingly, neither muscle-specific expression, autoregulation, or cross activation depends upon the presence of of these E-box or MEF-2 binding sites in the CMD1 promoter. These results demonstrate that the autoregulation and cross activation of the chicken MyoD promoter through the putative direct binding of the myogenic basic helix-loop-helix regulatory factors is mediated through an indirect pathway that involves unidentified regulatory elements and/or ancillary factors.

scholarly journals MMP activation–associated aminopeptidase N reveals a bivalent 14-3-3 binding motif

2020 ◽  
Vol 295 (52) ◽  
pp. 18266-18275
Author(s):  
Sebastian Kiehstaller ◽  
Christian Ottmann ◽  
Sven Hennig
Keyword(s):  
Binding Site ◽  
Binding Sites ◽  
Viral Infections ◽  
Binding Mode ◽  
Aminopeptidase N ◽  
Binding Motif ◽  
Adapter Proteins ◽  
Cellular Functions ◽  
Phosphorylated Peptides ◽  
Direct Binding

Aminopeptidase N (APN, CD13) is a transmembrane ectopeptidase involved in many crucial cellular functions. Besides its role as a peptidase, APN also mediates signal transduction and is involved in the activation of matrix metalloproteinases (MMPs). MMPs function in tissue remodeling within the extracellular space and are therefore involved in many human diseases, such as fibrosis, rheumatoid arthritis, tumor angiogenesis, and metastasis, as well as viral infections. However, the exact mechanism that leads to APN-driven MMP activation is unclear. It was previously shown that extracellular 14-3-3 adapter proteins bind to APN and thereby induce the transcription of MMPs. As a first step, we sought to identify potential 14-3-3–binding sites in the APN sequence. We constructed a set of phosphorylated peptides derived from APN to probe for interactions. We identified and characterized a canonical 14-3-3–binding site (site 1) within the flexible, structurally unresolved N-terminal APN region using direct binding fluorescence polarization assays and thermodynamic analysis. In addition, we identified a secondary, noncanonical binding site (site 2), which enhances the binding affinity in combination with site 1 by many orders of magnitude. Finally, we solved crystal structures of 14-3-3σ bound to mono- and bis-phosphorylated APN-derived peptides, which revealed atomic details of the binding mode of mono- and bivalent 14-3-3 interactions. Therefore, our findings shed some light on the first steps of APN-mediated MMP activation and open the field for further investigation of this important signaling pathway.

Molecular Modelling of the Interaction between DCMU and the QB-Binding Site of Photosystem II

1993 ◽  
Vol 48 (3-4) ◽  
pp. 191-198 ◽  
Author(s):  
Simon P. Mackay ◽  
Patrick J. O ’Malley
Keyword(s):  
Hydrogen Bond ◽  
Photosystem Ii ◽  
Binding Site ◽  
Binding Sites ◽  
The Other ◽  
Reaction Centre ◽  
Protein Model ◽  
Intermolecular Energy ◽  
Herbicide Binding ◽  
Binding Orientations

Abstract The prefered binding orientations for the herbicide DCMU within the QB-binding site of the D 1 protein model from a photosystem II reaction centre have been determined. Calculation of the intermolecular energy between the herbicide and the binding site has been instrumental in obtaining optimum positions reinforced by experimental results from mutation studies and herbicide binding to analogous bacterial reaction centres. We have shown that two binding sites are possible, one involving a hydrogen bond to and the other to the Ser 264 residue. In both cases, which are more important for the stabilization of the interactions.

THE PHOSPHOLIPID-BINDING SITE OF FACTOR VIII IS LOCATED ON THE 80 kD LIGHT CHAIN

1987 ◽  
Author(s):  
G Kemball-Cook ◽  
S J A Edwards ◽  
K Sewerin ◽  
L-O Andersson ◽  
T W Barrowcliffe
Keyword(s):  
Binding Site ◽  
Light Chain ◽  
Binding Sites ◽  
Immunoaffinity Chromatography ◽  
The Other ◽  
Immunological Methods ◽  
Electrophoretic Separation ◽  
Antigenic Sites ◽  
Reducing Conditions ◽  
Sds Page

The binding of Factoi. VIII (F.VIII) peptides to phospholipid (PL) vesicles has been studied by two different methods involving the use of fractionated anti-F.VIII:C I-Fab123’pre viously reported, i-Fab123’ was fractionated by immunoadsorptionwith F.VIII-PL complexes into two pools:one binding only to PL-binding sites on F.VIIIsAg (PL-site antibody), the other directed against other antigenic sites (non-PL-site antibody).The first technique used was a modification of the method of Weinstein et al. (Proc.Natl.Acad.Sci.USA, 78, 5137-5141, 1981), and involved incubation of the two anti-F.VIII pool swith F.VIII-containing samples, followed by electrophoretic separation of the complexes on the basis of size in non-denaturing SDS gels: this technique allows qualitative analysis of antibody reactive peptides in highly impure samples. Non-PL-site pool reacted with a range of peptides with MrMapparent Mr 90 kD up to 280 kD, a similar pattern to that of ’heavy chain’(HC) peptides of F.VIII seen on SDS-PAGE under reducing conditions; the PL-site antibody, however, reacted only with peptides at apparent Mrs of 80 kD and sometimes150 kD, but not with bands of higher Mr a pattern more consistent with binding to light chain (LC) peptides. Thesame patterns with the two labels were seen in both plasma and F.VIII concentrateThe second approach employed the two labels described above in direct immunoradiometric assays (IFMA’s) on purified human F.VIII peptides prepared by immunoaffinity chromatography and ion exchange on Mono Q gel. Both PL-site and non-PL-site labels measured similar amounts of F.VIII m a sample containing both HC and LC peptides; however, on assaying a sample containing purified HC peptides alone, PL-site antibody measured only 2% of F.VIII:Ag found by non-PL-site label, indicating that PL-binding sites present in samples containing both HC and LC are absent in HC alone.Results from both these immunological methods indicate that the 80 kD LC peptide of F.VIII carries the PL-binding site.

scholarly journals The actin filament-severing domain of plasma gelsolin.

1986 ◽  
Vol 103 (4) ◽  
pp. 1473-1481 ◽  
Author(s):  
C Chaponnier ◽  
P A Janmey ◽  
H L Yin
Keyword(s):  
Binding Site ◽  
Human Plasma ◽  
Actin Filament ◽  
Binding Sites ◽  
Actin Filaments ◽  
The Other ◽  
Actin Binding ◽  
Plasma Gelsolin ◽  
Chymotryptic Peptide ◽  
Native Plasma

Gelsolin, a multifunctional actin-modulating protein, has two actin-binding sites which may interact cooperatively. Native gelsolin requires micromolar Ca2+ for optimal binding of actin to both sites, and for expression of its actin filament-severing function. Recent work has shown that an NH2-terminal chymotryptic 17-kD fragment of human plasma gelsolin contains one of the actin-binding sites, and that this fragment binds to and severs actin filaments weakly irrespective of whether Ca2+ is present. The other binding site is Ca2+ sensitive, and is found in a chymotryptic peptide derived from the COOH-terminal two-thirds of plasma gelsolin; this fragment does not sever F-actin or accelerate the polymerization of actin. This paper documents that larger thermolysin-derived fragments encompassing the NH2-terminal half of gelsolin sever actin filaments as effectively as native plasma gelsolin, although in a Ca2+-insensitive manner. This result indicates that the NH2-terminal half of gelsolin is the actin-severing domain. The stringent Ca2+ requirement for actin severing found in intact gelsolin is not due to a direct effect of Ca2+ on the severing domain, but indirectly through an effect on domains in the COOH-terminal half of the molecule to allow exposure of both actin-binding sites.

scholarly journals A kinetic model for Ca2+ efflux mediated by the Ca2+ + Mg2+-activated ATPase of sarcoplasmic reticulum

1987 ◽  
Vol 245 (3) ◽  
pp. 713-721 ◽  
Author(s):  
J M McWhirter ◽  
G W Gould ◽  
J M East ◽  
A G Lee
Keyword(s):  
Experimental Data ◽  
Skeletal Muscle ◽  
Sarcoplasmic Reticulum ◽  
Kinetic Model ◽  
Atpase Activity ◽  
Binding Site ◽  
Binding Sites ◽  
Adenine Nucleotides ◽  
Contraction Coupling ◽  
High Affinity

We present a model for Ca2+ efflux from vesicles of sarcoplasmic reticulum (SR). It is proposed that efflux is mediated by the Ca2+ + Mg2+-activated ATPase that is responsible for Ca2+ uptake in this system. In the normal ATPase cycle of the ATPase, phosphorylation of the ATPase is followed by a conformational change in which the Ca2+-binding sites change from being outward-facing and of high affinity to being inward-facing and of low affinity. To mediate Ca2+ efflux, it is proposed that the ATPase can adopt a conformation in which the Ca2+-binding sites are of low affinity but still outward-facing. It is shown that experimental data on the rates of Ca2+ efflux can be simulated in terms of this model, with Ca2+-binding-site affinities previously proposed to explain ATPase activity [Gould, East, Froud, McWhirter, Stefanova & Lee (1986) Biochem. J. 237, 217-227]. Effects of Mg2+ and adenine nucleotides on efflux rates are explained. It is suggested that Ca2+ efflux from SR mediated by the ATPase could be important in excitation-contraction coupling in skeletal muscle.

scholarly journals E-box- and MEF-2-independent muscle-specific expression, positive autoregulation, and cross-activation of the chicken MyoD (CMD1) promoter reveal an indirect regulatory pathway.

1994 ◽  
Vol 14 (8) ◽  
pp. 5474-5486 ◽  
Author(s):  
C A Dechesne ◽  
Q Wei ◽  
J Eldridge ◽  
L Gannoun-Zaki ◽  
P Millasseau ◽  
...  
Keyword(s):  
Binding Site ◽  
Binding Sites ◽  
Primary Cultures ◽  
Regulatory Elements ◽  
The Other ◽  
Indirect Pathway ◽  
Specific Expression ◽  
Fibroblast Cultures ◽  
E Box ◽  
Myogenic Factors

Members of the MyoD family of gene-regulatory proteins (MyoD, myogenin, myf5, and MRF4) have all been shown not only to regulate the transcription of numerous muscle-specific genes but also to positively autoregulate and cross activate each other's transcription. In the case of muscle-specific genes, this transcriptional regulation can often be correlated with the presence of a DNA consensus in the regulatory region CANNTG, known as an E box. Little is known about the regulatory interactions of the myogenic factors themselves; however, these interactions are thought to be important for the activation and maintenance of the muscle phenotype. We have identified the minimal region in the chicken MyoD (CMD1) promoter necessary for muscle-specific transcription in primary cultures of embryonic chicken skeletal muscle. The CMD1 promoter is silent in primary chick fibroblast cultures and in muscle cell cultures treated with the thymidine analog bromodeoxyuridine. However, CMD1 and chicken myogenin, as well as, to a lesser degree, chicken Myf5 and MRF4, expressed in trans can activate transcription from the minimal CMD1 promoter in these primary fibroblast cultures. Here we show that the CMD1 promoter contains numerous E-box binding sites for CMD1 and the other myogenic factors, as well as a MEF-2 binding site. Surprisingly, neither muscle-specific and the other myogenic factors, as well as a MEF-2 binding site. Surprisingly, neither muscle-specific expression, autoregulation, or cross activation depends upon the presence of of these E-box or MEF-2 binding sites in the CMD1 promoter. These results demonstrate that the autoregulation and cross activation of the chicken MyoD promoter through the putative direct binding of the myogenic basic helix-loop-helix regulatory factors is mediated through an indirect pathway that involves unidentified regulatory elements and/or ancillary factors.

scholarly journals Regulation of Vps4 ATPase activity by ESCRT-III

2009 ◽  
Vol 37 (1) ◽  
pp. 143-145 ◽  
Author(s):  
Brian A. Davies ◽  
Ishara F. Azmi ◽  
David J. Katzmann
Keyword(s):  
Atpase Activity ◽  
Multivesicular Body ◽  
Critical Function ◽  
Body Formation ◽  
Temporal Regulation ◽  
Endosomal Sorting ◽  
Aaa Atpase ◽  
Endosomal Membrane ◽  
Endosomal Membranes ◽  
Vacuolar Protein

MVB (multivesicular body) formation occurs when the limiting membrane of an endosome invaginates into the intraluminal space and buds into the lumen, bringing with it a subset of transmembrane cargoes. Exvagination of the endosomal membrane from the cytosol is topologically similar to the budding of retroviral particles and cytokinesis, wherein membranes bud away from the cytoplasm, and the machinery responsible for MVB sorting has been implicated in these phenomena. The AAA (ATPase associated with various cellular activities) Vps4 (vacuolar protein sorting 4) performs a critical function in the MVB sorting pathway. Vps4 appears to dissociate the ESCRTs (endosomal sorting complexes required for transport) from endosomal membranes during the course of MVB sorting, but it is unclear how Vps4 ATPase activity is synchronized with ESCRT release. We have investigated the mechanisms by which ESCRT components stimulate the ATPase activity of Vps4. These studies support a model wherein Vps4 activity is subject to spatial and temporal regulation via distinct mechanisms during MVB sorting.

Specific Labelling of the Curare Binding Sites of Acetylcholinesterase and Some Properties of the Modified Enzyme

1971 ◽  
Vol 49 (10) ◽  
pp. 1131-1133 ◽  
Author(s):  
B. Belleau ◽  
V. DiTullio
Keyword(s):  
Binding Site ◽  
Binding Sites ◽  
The Other ◽  
Excitable Membranes ◽  
Electric Eel ◽  
Modified Enzyme ◽  
Enzyme Species ◽  
Specific Labelling

Electric eel acetylcholinesterase was found to selectively react covalently with two molecules of labelled N,N-dimethyl-2-phenylaziridinium chloride (14C-DPA) per unit of 65 000 daltons. One molecule of bound DPA is selectively labilized at pH 9.5. Both alkylated enzyme species (E.2DPA and E.DPA) are inactive toward acetylcholine as substrate, but considerably more reactive toward indophenylacetate (IPA) as substrate. The IPA activities of both the E.2DPA and E.DPA enzymes are unaffected by decamethonium bromide (C10), whereas only the latter suffers inhibition by d-tubocurarine. It is concluded that the enzyme moiety where quaternary effectors interact possesses two topographically distinct classes of sites: one class is essential for C10 inhibition of the esteratic activity, and the other would serve as an allosteric binding site for curare. This latter site may play the role of receptor on excitable membranes.

scholarly journals Anti-phosphocholine hybridoma antibodies. II. Functional analysis of binding sites within three antibody families.

1981 ◽  
Vol 154 (5) ◽  
pp. 1584-1598 ◽  
Author(s):  
C M Andres ◽  
A Maddalena ◽  
S Hudak ◽  
N M Young ◽  
J L Claflin
Keyword(s):  
Binding Site ◽  
Binding Sites ◽  
Structural Heterogeneity ◽  
The Other ◽  
Family Diversity ◽  
Differential Reactivity ◽  
Myeloma Proteins ◽  
Rational Explanation ◽  
Intra Family

The present investigation extends our immunochemical characterization of binding site heterogeneity among a large series of monoclonal anti-phosphocholine (PC) antibodies. Hybridoma proteins (HP) from eight genetically distinct strains are included in this study, yet no strain specific characteristics are observed. These HP, as previously shown (5), are divided into three well-defined families based on public or family-specific Id and L chain isotypes characteristic of three PC-binding myeloma proteins: T15, M603, and M511. All antibodies exhibited some degree of inter- or intra-family heterogeneity, or both. Some of this intra-family diversity was reflected by differential reactivity for PC when attached to three different carriers. In spite of this, the specificity profiles for hapten analogues of PC, as measured by hapten inhibition of binding, were the same for all members of the T15 family. Altering the carrier had no effect, thus suggesting that the binding site pocket for PC is essentially preserved, whereas that for carrier is variable. Similar conclusions were reached for most of the M603 HP, although the binding site is different from the T15 HP. The M511 HP stand in sharp contrast to the HP in the other two families because their binding sites exhibit extensive variability. The independence in reactivity for PC and PC plus carrier offers a rational explanation for idiotypic and/or structural heterogeneity within a family. More importantly it suggests interesting strategies for diversification within one group of antibodies.

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