scholarly journals Co-Immunoprecipitation Reveals Interactions Between Amelogenin and Ameloblastin via Their Self-Assembly Domains

2020 ◽  
Vol 11 ◽  
Author(s):  
Rucha Arun Bapat ◽  
Jingtan Su ◽  
Janet Moradian-Oldak
Keyword(s):  
Self Assembly ◽  
Dental Enamel ◽  
Enamel Matrix Proteins ◽  
Enamel Formation ◽  
Macromolecular Assembly ◽  
Show Evidence ◽  
Maturation Stages ◽  
Mouse Incisor

Macromolecular assembly of extracellular enamel matrix proteins (EMPs) is intimately associated with the nucleation, growth, and maturation of highly organized hydroxyapatite crystals giving rise to healthy dental enamel. Although the colocalization of two of the most abundant EMPs amelogenin (Amel) and ameloblastin (Ambn) in molar enamel has been established, the evidence toward their interaction is scarce. We used co-immunoprecipitation (co-IP) to show evidence of direct molecular interactions between recombinant and native Amel and Ambn. Ambn fragments containing Y/F-x-x-Y/L/F-x-Y/F self-assembly motif were isolated from the co-IP column and characterized by mass spectroscopy. We used recombinant Ambn (rAmbn) mutants with deletion of exons 5 and 6 as well as Ambn derived synthetic peptides to demonstrate that Ambn binds to Amel via its previously identified Y/F-x-x-Y/L/F-x-Y/F self-assembly motif at the N-terminus of its exon 5 encoded region. Using an N-terminal specific anti-Ambn antibody, we showed that Ambn N-terminal fragments colocalized with Amel from secretory to maturation stages of enamel formation in a single section of developing mouse incisor, and closely followed mineral patterns in enamel rod interrod architecture. We conclude that Ambn self-assembly motif is involved in its interaction with Amel in solution and that colocalization between the two proteins persists from secretory to maturation stages of amelogenesis. Our in vitro and in situ data support the notion that Amel and Ambn may form heteromolecular assemblies that may perform important physiological roles during enamel formation.

Perturbed Amelogenin Protein Self-assembly Alters Nanosphere Properties Resulting in Defective Enamel Formation

2004 ◽  
Vol 823 ◽  
Author(s):  
Michael L. Paine ◽  
YaPing Lei ◽  
Wen Luo ◽  
Malcolm L. Snead
Keyword(s):  
Amino Acid ◽  
Self Assembly ◽  
Recombinant Dna ◽  
Point Mutations ◽  
Dental Enamel ◽  
Crystal Habit ◽  
Single Amino Acid ◽  
Enamel Formation ◽  
Amino Terminal

AbstractDental enamel is a unique composite bioceramic material that is the hardest tissue in the vertebrate body, containing long-, thin-crystallites of substituted hydroxyapatite. Enamel functions under immense loads in a bacterial-laden environment, and generally without catastrophic failure over a lifetime for the organism. Unlike all other biogenerated hard tissues of mesodermal origin, such as bone and dentin, enamel is produced by ectoderm-derived cells called ameloblasts. Recent investigations on the formation of enamel using cell and molecular approaches have been coupled to biomechanical investigations at the nanoscale and mesoscale levels. For amelogenin, the principle protein of forming enamel, two domains have been identified that are required for the proper assembly of multimeric units of amelogenin to form nanospheres. One domain is at the amino-terminus and the other domain in the carboxyl-terminal region. Amelogenin nanospheres are believed to influence the hydroxyapatite crystal habit. Both the yeast two-hybrid assay and surface plasmon resonance have been used to examine the assembly properties of engineered amelogenin proteins. Amelogenin protein was engineered using recombinant DNA techniques to contain deletions to either of the two self-assembly domains. Amelogenin protein was also engineered to contain single amino-acid mutations/substitutions in the amino-terminal self-assembly domain; and these amino-acid changes are based upon point mutations observed in humans affected with a hereditary disturbance of enamel formation. All of these alterations reveal significant defects in amelogenin self-assembly into nanospheres in vitro. Transgenic animals containing these same amelogenin deletions illustrate the importance of a physiologically correct bio-fabrication of the enamel protein extracellular matrix to allow for the organization of the enamel prismatic structure.

Crystal Growth in Dental Enamel: the Role of Amelogenins and Albumin

1996 ◽  
Vol 10 (2) ◽  
pp. 173-180 ◽  
Author(s):  
C. Robinson ◽  
S.J. Brookes ◽  
J. Kirkham ◽  
W.A. Bonass ◽  
R.C. Shore
Keyword(s):  
Crystal Growth ◽  
Phosphate Buffer ◽  
Significant Proportion ◽  
Dental Enamel ◽  
Maturation Stage ◽  
Enamel Matrix Proteins ◽  
Rat Incisor ◽  
Transition Stage ◽  
Maturation Stages

Amelogenin-mineral interactions were investigated using an in vitro binding approach. Rat incisor enamel matrix proteins (mainly amelogenins) were dissolved in synthetic enamel fluid and allowed to equilibrate with deproteinised developing enamel crystals. The results showed that amlogenin proteins of 21, 23, 24, 26 and 27-kDa (corresponding to nascent and partially degraded amelogenins) were associated with the crystals whilst the lower Mr amelogenins (<21KDa) remained free in the synthetic enamel fluid. These data suggest the nascent and partially degraded amelogenins may interact with developing enamel crystals and could influence their growth. Albumin-mineral interactions were investigated by extracting developing rat incisor enamel with synthetic enamel fluid. Insoluble material (including the enamel crystals) was then further extracted with 0.1 M phosphate buffer (pH 7.4) to desorb any mineral bound proteins. Western blotting using anti-albumin antibodies showed that almost all of the albumin from the secretory stage enamel and a significant proportion of the albumin present in early transition stage was extractable in the synthetic enamel fluid. However. synthetic enamel fluid did not extract albumin from late transition or maturation stage tissue, which could only be removed following further extraction with phosphate buffer. Albumin degradation was apparent during the transition and maturation stages, where it is degraded and ultimately removed. This binding pattern may be related to amelogenin degradation and removal during the transition stage,. permitting albumin access to the previously obscured crystal surfaces. That the secretory stage matrix appears to "protect" secretory stage crystals from albumin may be an important consideration in the aetiology of enamel hypoplasias (i.e. incomplete crystal growth) and when using dissociative extraction procedures for the identification of mineral bound proteins.

In vitro and in vivo polysaccharides assembly: ultrastructural and cytochemical study of growing plant cell wall components.

1978 ◽  
Vol 36 (2) ◽  
pp. 434-435
Author(s):  
D. Reis ◽  
B. Vian ◽  
J. C. Roland
Keyword(s):  
Cell Wall ◽  
Self Assembly ◽  
Plant Cell Wall ◽  
Higher Plants ◽  
Three Dimensional ◽  
Cytochemical Study ◽  
Wall Components

Wall morphogenesis in higher plants is a problem still open to controversy. Until now the possibility of a transmembrane control and the involvement of microtubules were mostly envisaged. Self-assembly processes have been observed in the case of walls of Chlamydomonas and bacteria. Spontaneous gelling interactions between xanthan and galactomannan from Ceratonia have been analyzed very recently. The present work provides indications that some processes of spontaneous aggregation could occur in higher plants during the formation and expansion of cell wall.Observations were performed on hypocotyl of mung bean (Phaseolus aureus) for which growth characteristics and wall composition have been previously defined.In situ, the walls of actively growing cells (primary walls) show an ordered three-dimensional organization (fig. 1). The wall is typically polylamellate with multifibrillar layers alternately transverse and longitudinal. Between these layers intermediate strata exist in which the orientation of microfibrils progressively rotates. Thus a progressive change in the morphogenetic activity occurs.

scholarly journals Development of In Situ Self-Assembly Nanoparticles to Encapsulate Lopinavir and Ritonavir for Long-Acting Subcutaneous Injection

Pharmaceutics ◽  
2021 ◽  
Vol 13 (6) ◽  
pp. 904
Author(s):  
Irin Tanaudommongkon ◽  
Asama Tanaudommongkon ◽  
Xiaowei Dong
Keyword(s):  
Sustained Release ◽  
Trough Concentration ◽  
Self Assembly ◽  
Subcutaneous Injection ◽  
Drug Loading ◽  
Entrapment Efficiency ◽  
Long Acting

Most antiretroviral medications for human immunodeficiency virus treatment and prevention require high levels of patient adherence, such that medications need to be administered daily without missing doses. Here, a long-acting subcutaneous injection of lopinavir (LPV) in combination with ritonavir (RTV) using in situ self-assembly nanoparticles (ISNPs) was developed to potentially overcome adherence barriers. The ISNP approach can improve the pharmacokinetic profiles of the drugs. The ISNPs were characterized in terms of particle size, drug entrapment efficiency, drug loading, in vitro release study, and in vivo pharmacokinetic study. LPV/RTV ISNPs were 167.8 nm in size, with a polydispersity index of less than 0.35. The entrapment efficiency was over 98% for both LPV and RTV, with drug loadings of 25% LPV and 6.3% RTV. A slow release rate of LPV was observed at about 20% on day 5, followed by a sustained release beyond 14 days. RTV released faster than LPV in the first 5 days and slower than LPV thereafter. LPV trough concentration remained above 160 ng/mL and RTV trough concentration was above 50 ng/mL after 6 days with one subcutaneous injection. Overall, the ISNP-based LPV/RTV injection showed sustained release profiles in both in vitro and in vivo studies.

Protein self-assembly following in situ expression in artificial and mammalian cells

2017 ◽  
Vol 9 (5) ◽  
pp. 444-450 ◽  
Author(s):  
Urszula M. Migas ◽  
Michelle K. Quinn ◽  
Jennifer J. McManus
Keyword(s):  
Self Assembly ◽  
Mammalian Cells

The importance of in vitro measurements in explaining the mechanisms underlying protein self-assembly in physiologically relevant conditions has been demonstrated in solution and in artificial and mammalian cells.

Structure-Property Correlation in Genetically-Engineered Mouse Enamel

2001 ◽  
Vol 7 (S2) ◽  
pp. 992-993
Author(s):  
Hanson Fong ◽  
Daniel Heidel ◽  
Mehmet Sarikaya ◽  
Michael Paine ◽  
Wen Lou ◽  
...  
Keyword(s):  
Self Assembly ◽  
Matrix Protein ◽  
Dental Enamel ◽  
Genetically Engineered ◽  
Structure Property ◽  
Biomineralization Process ◽  
Enamel Epithelium ◽  
Assembly Behavior

Dental enamel is the most durable bioceramics produced by a vertebrate as it is designed to perform masticatory functions throughout its lifetime. The understanding of the mechanism of enamel formation and effects of proteins during the biomineralization process are fundamental issues, essential for both potential enamel regeneration and as a base for synthesis, via self-assembly, of biomimetic composites.The biomineralization process of enamel is carried out by ameloblast cells that line the inner enamel epithelium and secrete an extracellular protein matrix onto a mineralized dentin surface at the dentin-enamel junction (DEJ). A major matrix protein, amelogenin, is believed to regulate the mineralization of hydroxyapatite (HAP) in the enamel tissue. It has been shown to undergo self-assembly in vitro and in vivo to form nanospheres of ∼20nm in diameter. Previous TEM studies have shown that the nanospheres align along the length (c-axis) of hydroxyapatite (HA) crystals. There are two domains, namely A (residues 1-42) and B (residues 157-173), that control the self-assembly behavior of the nanospheres.

scholarly journals Enzymatic processing of amelogenin during continuous crystallization of apatite

2008 ◽  
Vol 23 (12) ◽  
pp. 3184-3195 ◽  
Author(s):  
V. Uskoković ◽  
M-K. Kim ◽  
W. Li ◽  
S. Habelitz
Keyword(s):  
Self Assembly ◽  
Weight Ratio ◽  
Dental Enamel ◽  
Degradation Process ◽  
Continuous Crystallization ◽  
Phosphate Ions ◽  
Ph Stat ◽  
In Vitro Effects ◽  
Titration System

Dental enamel forms through a protein-controlled mineralization and enzymatic degradation process with a nanoscale precision that new engineering technologies may be able to mimic. Recombinant full-length human amelogenin (rH174) and a matrix-metalloprotease (MMP-20) were used in a pH-stat titration system that enabled a continuous supply of calcium and phosphate ions over several days, mimicking the initial stages of matrix processing and crystallization in enamel in vitro. Effects on the self-assembly and crystal growth from a saturated aqueous solution containing 0.4 mg/mL rH174 and MMP-20 with the weight ratio of 1:1000 with respect to rH174 were investigated. A transition from nanospheres to fibrous amelogenin assemblies was facilitated under conditions that involved interaction between rH174 and its proteolytic cleavage products. Despite continuous titration, the levels of calcium exhibited a consistent trend of decreasing, thereby indicating a possible role in protein self-assembly. This study suggests that mimicking enamel formation in vitro requires the synergy between the aspects of matrix self-assembly, proteolysis, and crystallization.

The Effect of Copper on Demineralization of Dental Enamel

2006 ◽  
Vol 85 (11) ◽  
pp. 1011-1015 ◽  
Author(s):  
A.Z. Abdullah ◽  
S.M. Strafford ◽  
S.J. Brookes ◽  
M.S. Duggal
Keyword(s):  
Dental Enamel ◽  
Double Blind ◽  
Fluoride Treatment ◽  
Before And After ◽  
Amine Fluoride ◽  
Knoop Microhardness ◽  
Effect Of Copper ◽  
Removable Appliance

Previous studies have concluded that copper might inhibit enamel demineralization in vitro. Our aim was to assess the effect of copper (Cu2+), with and without amine fluoride, on human dental enamel under cariogenic challenge in situ. In a double-blind randomized four-leg crossover trial, 14 individuals wore a removable appliance containing 2 enamel slabs, 1 containing an artificial caries lesion. During each leg, the appliance was exposed twice daily to one of the test solutions: 1.25 mM CuSO4, amine fluoride (250 ppm F), copper and amine fluoride combined, or a placebo (water). A cariogenic challenge was provided in all cases by 5 daily exposures to 10% sucrose. Slabs were assessed before and after 21 days’ exposure by Knoop microhardness and transverse microradiography. Significantly less demineralization was observed with Cu2+ and fluoride in combination than with fluoride treatment alone (p < 0.05), whereas copper alone had no significant protective effect.

Effect of FKBP65, a putative elastin chaperone, on the coacervation of tropoelastin in vitro

2010 ◽  
Vol 88 (6) ◽  
pp. 917-925 ◽  
Author(s):  
Kevin L.Y. Cheung ◽  
Matthew Bates ◽  
Vettai S. Ananthanarayanan
Keyword(s):  
Self Assembly ◽  
Secretory Pathway ◽  
Molar Ratio ◽  
Prolyl Isomerase ◽  
Ppiase Activity ◽  
Peptidyl Prolyl Isomerase ◽  
Maturation Stages ◽  
Turbidimetric Assay

FKBP65 is a protein of the endoplasmic reticulum that is relatively abundant in elastin-producing cells and is associated with tropoelastin in the secretory pathway. To test an earlier suggestion by Davis and co-workers that FKBP65 could act as an intracellular chaperone for elastin, we obtained recombinant FKBP65 (rFKBP65) by expressing it in E. coli and examined its effect on the coacervation characteristics of chicken aorta tropoelastin (TE) using an in vitro turbidimetric assay. Our results reveal that rFKBP65 markedly promotes the initiation of coacervation of TE without significantly affecting the temperature of onset of coacervation. This effect shows saturation at a 1:2 molar ratio of TE to rFKBP65. By contrast, FKBP12, a peptidyl prolyl isomerase, has a negligible effect on TE coacervation. Moreover, the effect of rFKBP65 on TE coacervation is unaffected by the addition of rapamycin, an inhibitor of peptidyl prolyl isomerase (PPIase) activity. These observations rule out the involvement of the PPIase activity of rFKBP65 in modulating the coacervation of TE. Additional experiments using a polypeptide model of TE showed that rFKBP65, while promoting coacervation, may retard the maturation of this model polypeptide into larger aggregates. Based on these results, we suggest that FKBP65 may act as an elastin chaperone in vivo by controlling both the coacervation and the maturation stages of its self-assembly into fibrils.

Role of amelogenin self-assembly in protein-mediated dental enamel formation

2010 ◽  
pp. 369-374
Author(s):  
Henry C. Margolis ◽  
Felicitas B. Wiedemann-Bidlack ◽  
Barbara Aichmayer ◽  
Peter Fratzl ◽  
Seo-Young Kwak ◽  
...  
Keyword(s):  
Self Assembly ◽  
Dental Enamel ◽  
Enamel Formation
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