Effects of phosphorylation on the self-assembly of native full-length porcine amelogenin and its regulation of calcium phosphate formation in vitro

AbstractA novel concept has emerged whereby the higher-order self-assembly of proteins provides a simple and robust mechanism for signal amplification. This appears to be a universal signalling mechanism within the innate immune system, where the recognition of pathogens or danger-associated molecular patterns need to trigger a strong, binary response within cells. Previously, multiple structural studies have been limited to single domains, expressed and assembled at high protein concentrations. We therefore set out to develop new in vitro strategies to characterise the behaviour of full-length proteins at physiological levels. In this study we focus on the adaptor protein MyD88, which contains two domains with different self-assembly properties: a TIR domain that can polymerise similarly to the TIR domain of Mal, and a Death Domain that has been shown to oligomerise with helical symmetry in the Myddosome complex. To visualize the behaviour of full-length MyD88 without purification steps, we use single-molecule fluorescence coupled to eukaryotic cell-free protein expression. These experiments demonstrate that at low protein concentration, only full-length MyD88 forms prion-like polymers. We also demonstrate that the metastability of MyD88 polymerisation creates the perfect binary response required in innate signalling: the system is silenced at normal concentrations but upstream signalling creates a “seed” that triggers polymerisation and amplification of the response. These findings pushed us to re-interpret the role of polymerisation in MyD88-related diseases and we studied the impact of disease-associated point mutations L93P, R196C and L252P/L265P at the molecular level. We discovered that all mutations completely block the ability of MyD88 to polymerise. We also confirm that L252P, a gain-of-function mutation, allows the MyD88 mutant to form extremely stable oligomers, even when expressed at low nanomolar concentrations. Thus, our results are consistent with and greatly add to the findings on the Myddosomes digital ‘all-or-none’ responses and the behaviour of the oncogenic mutation of MyD88.

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Biochemical reconstitutions reveal principles of human γ-TuRC assembly and function

The Journal of Cell Biology ◽

10.1083/jcb.202009146 ◽

2021 ◽

Vol 220 (3) ◽

Cited By ~ 1

Author(s):

Michal Wieczorek ◽

Shih-Chieh Ti ◽

Linas Urnavicius ◽

Kelly R. Molloy ◽

Amol Aher ◽

...

Keyword(s):

Electron Microscopy ◽

Self Assembly ◽

The Self ◽

Dependent Manner ◽

Guanine Nucleotide ◽

Partial Cone ◽

Ring Complex ◽

Microtubule Networks ◽

And Function

The formation of cellular microtubule networks is regulated by the γ-tubulin ring complex (γ-TuRC). This ∼2.3 MD assembly of >31 proteins includes γ-tubulin and GCP2-6, as well as MZT1 and an actin-like protein in a “lumenal bridge” (LB). The challenge of reconstituting the γ-TuRC has limited dissections of its assembly and function. Here, we report a biochemical reconstitution of the human γ-TuRC (γ-TuRC-GFP) as a ∼35 S complex that nucleates microtubules in vitro. In addition, we generate a subcomplex, γ-TuRCΔLB-GFP, which lacks MZT1 and actin. We show that γ-TuRCΔLB-GFP nucleates microtubules in a guanine nucleotide–dependent manner and with similar efficiency as the holocomplex. Electron microscopy reveals that γ-TuRC-GFP resembles the native γ-TuRC architecture, while γ-TuRCΔLB-GFP adopts a partial cone shape presenting only 8–10 γ-tubulin subunits and lacks a well-ordered lumenal bridge. Our results show that the γ-TuRC can be reconstituted using a limited set of proteins and suggest that the LB facilitates the self-assembly of regulatory interfaces around a microtubule-nucleating “core” in the holocomplex.

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MMP20 Proteolysis of Native Amelogenin Regulates Mineralization In Vitro

Journal of Dental Research ◽

10.1177/0022034516662814 ◽

2016 ◽

Vol 95 (13) ◽

pp. 1511-1517 ◽

Cited By ~ 14

Author(s):

S.Y. Kwak ◽

Y. Yamakoshi ◽

J.P. Simmer ◽

H.C. Margolis

Keyword(s):

Calcium Phosphate ◽

Full Length ◽

Protein Solutions ◽

Mineral Formation ◽

C Protein ◽

Ordered Arrays ◽

Transmission Electron ◽

Precursor Phase ◽

Lower Capacity

Recent studies have shown that native phosphorylated full-length porcine amelogenin (P173) and its predominant cleavage product (P148) can inhibit spontaneous calcium phosphate formation in vitro by stabilizing an amorphous calcium phosphate (ACP) precursor phase. Since full-length amelogenin undergoes proteolysis by matrix metalloproteinase 20 (MMP20, enamelysin) soon after secretion, the present study was conducted to assess the effect of amelogenin proteolysis on calcium phosphate formation. Calcium and phosphate were sequentially added to protein solutions without and with added MMP20 (ratio = 200:1) under physiological-like conditions of ionic strength (163 mM) in 50 mM Tris-HCl (pH 7.4) at 37 °C. Protein degradation with time was assessed by gel-electrophoresis, and mineral products formed were characterized by transmission electron microscopy (TEM). MMP20 was found to cleave P173 to primarily generate P148, along with P162, P46-148, and P63/64-148. In sharp contrast, MMP20 did not cleave P148. In addition, the formation of well-aligned bundles of enamel-like hydroxyapatite (HA) crystals was promoted in the presence of P173 with added MMP20, while only ACP particles were seen in the absence of MMP20. Although P148 was found to have a somewhat lower capacity to stabilize ACP and prevent HA formation compared with P173 in the absence of MMP20, essentially no HA formation was observed in the presence of somewhat higher concentrations of P148 regardless of MMP20 addition, due to the lack of observed protein proteolysis. Present findings suggest that ACP transformation to ordered arrays of enamel crystals may be regulated in part by the proteolysis of full-length native amelogenin, while the predominant amelogenin degradation product in developing enamel (e.g., P148) primarily serves to prevent uncontrolled mineral formation during the secretory stage of amelogenesis.

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Reconstitution of selective HIV-1 RNA packaging in vitro by membrane-bound Gag assemblies

eLife ◽

10.7554/elife.14663 ◽

2016 ◽

Vol 5 ◽

Cited By ~ 21

Author(s):

Lars-Anders Carlson ◽

Yun Bai ◽

Sarah C Keane ◽

Jennifer A Doudna ◽

James H Hurley

Keyword(s):

Self Assembly ◽

Untranslated Region ◽

Minimal System ◽

The Self ◽

Viral Rna ◽

Large Excess ◽

Rna Packaging ◽

Membrane Bound ◽

Hiv 1

HIV-1 Gag selects and packages a dimeric, unspliced viral RNA in the context of a large excess of cytosolic human RNAs. As Gag assembles on the plasma membrane, the HIV-1 genome is enriched relative to cellular RNAs by an unknown mechanism. We used a minimal system consisting of purified RNAs, recombinant HIV-1 Gag and giant unilamellar vesicles to recapitulate the selective packaging of the 5’ untranslated region of the HIV-1 genome in the presence of excess competitor RNA. Mutations in the CA-CTD domain of Gag which subtly affect the self-assembly of Gag abrogated RNA selectivity. We further found that tRNA suppresses Gag membrane binding less when Gag has bound viral RNA. The ability of HIV-1 Gag to selectively package its RNA genome and its self-assembly on membranes are thus interdependent on one another.

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Reconstructed Skin by the Self‐Assembly Approach: Is the Stem Cell Niche Present In Vitro?

The FASEB Journal ◽

10.1096/fasebj.22.1_supplement.522.7 ◽

2008 ◽

Vol 22 (S1) ◽

Author(s):

Amélie Lavoie ◽

Paquet Claudie ◽

Danielle Larouche ◽

Claudia Fugère ◽

Israel Martel ◽

...

Keyword(s):

Stem Cell ◽

Self Assembly ◽

Stem Cell Niche ◽

The Self ◽

Reconstructed Skin ◽

Cell Niche

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Biophysical and biochemical properties of Deup1 self-assemblies: a potential driver for deuterosome formation during multiciliogenesis

Biology Open ◽

10.1242/bio.056432 ◽

2021 ◽

Vol 10 (3) ◽

pp. bio056432 ◽

Cited By ~ 1

Author(s):

Shohei Yamamoto ◽

Ryoichi Yabuki ◽

Daiju Kitagawa

Keyword(s):

Self Assembly ◽

Large Scale ◽

Molecular Mechanisms ◽

Coiled Coil ◽

Biochemical Properties ◽

The Self ◽

Centrosomal Protein ◽

Biochemical Analyses ◽

Stable Structures

ABSTRACTThe deuterosome is a non-membranous organelle involved in large-scale centriole amplification during multiciliogenesis. Deuterosomes are specifically assembled during the process of multiciliogenesis. However, the molecular mechanisms underlying deuterosome formation are poorly understood. In this study, we investigated the molecular properties of deuterosome protein 1 (Deup1), an essential protein involved in deuterosome assembly. We found that Deup1 has the ability to self-assemble into macromolecular condensates both in vitro and in cells. The Deup1-containing structures formed in multiciliogenesis and the Deup1 condensates self-assembled in vitro showed low turnover of Deup1, suggesting that Deup1 forms highly stable structures. Our biochemical analyses revealed that an increase of the concentration of Deup1 and a crowded molecular environment both facilitate Deup1 self-assembly. The self-assembly of Deup1 relies on its N-terminal region, which contains multiple coiled coil domains. Using an optogenetic approach, we demonstrated that self-assembly and the C-terminal half of Deup1 were sufficient to spatially compartmentalize centrosomal protein 152 (Cep152) and polo like kinase 4 (Plk4), master components for centriole biogenesis, in the cytoplasm. Collectively, the present data suggest that Deup1 forms the structural core of the deuterosome through self-assembly into stable macromolecular condensates.This article has an associated First Person interview with the first author of the paper.

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Human Organ-Specific 3D Cancer Models Produced by the Stromal Self-Assembly Method of Tissue Engineering for the Study of Solid Tumors

BioMed Research International ◽

10.1155/2020/6051210 ◽

2020 ◽

Vol 2020 ◽

pp. 1-23 ◽

Cited By ~ 1

Author(s):

Vincent Roy ◽

Brice Magne ◽

Maude Vaillancourt-Audet ◽

Mathieu Blais ◽

Stéphane Chabaud ◽

...

Keyword(s):

Tissue Engineering ◽

Tumor Microenvironment ◽

Self Assembly ◽

The Self ◽

Human Organ ◽

Assembly Method ◽

Cancer Models ◽

Organ Specific

Cancer research has considerably progressed with the improvement of in vitro study models, helping to understand the key role of the tumor microenvironment in cancer development and progression. Over the last few years, complex 3D human cell culture systems have gained much popularity over in vivo models, as they accurately mimic the tumor microenvironment and allow high-throughput drug screening. Of particular interest, in vitrohuman 3D tissue constructs, produced by the self-assembly method of tissue engineering, have been successfully used to model the tumor microenvironment and now represent a very promising approach to further develop diverse cancer models. In this review, we describe the importance of the tumor microenvironment and present the existing in vitro cancer models generated through the self-assembly method of tissue engineering. Lastly, we highlight the relevance of this approach to mimic various and complex tumors, including basal cell carcinoma, cutaneous neurofibroma, skin melanoma, bladder cancer, and uveal melanoma.

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Polymorphism of tubulin assembly in vitro

Journal of Cell Science ◽

10.1242/jcs.94.3.479 ◽

1989 ◽

Vol 94 (3) ◽

pp. 479-488

Author(s):

PAULA KARECLA ◽

ELIZABETH HIRST ◽

PETER BAYLEY

Keyword(s):

Self Assembly ◽

Basic Structure ◽

Limited Range ◽

The Self ◽

Tubulin Dimer ◽

Thin Sectioning ◽

Associated Proteins ◽

Bona Fide ◽

Polymorphic Forms

Polymorphism in the self-assembly of tubulin dimer and microtubule protein (tubulin plus the microtubule-associated proteins) has been investigated as a function of systematic variation of solution composition (i.e. buffer ion, [glycerol] and [Mg2+]). The nature of the assembly product was examined using negative staining and thin sectioning electron microscopy. The morphology of the product of assembly of tubulin dimer was found to be strongly influenced by the concentration of glycerol and Mg2+ in Pipes and Mes buffers; the effects are less marked in phosphate buffer. Formation of bona fide microtubules in O.l M-Pipes occurs for a limited range of solution conditions (e.g. with [glycerol] <2 M and [Mg2+]<1mM). Conditions of elevated [glycerol] and [Mg2+], which enhance the rate and extent of assembly, have the adverse effect of strongly promoting the formation of polymorphic forms in addition to, and in place of, the normal microtubule morphology. In both Pipes and Mes buffers, increasing [glycerol] from 1 to 3 M favours the formation of extended multiply curved sheets, apparently made up from a basic structure with an S-like crosssection. By contrast, increasing [Mg2+] promotes the formation of junctions between microtubule walls, giving products whose cross-section shows multiple hook-like appendages, attached to closed microtubules. The assembly of tubulin dimer in a typical ‘dimer assembly buffer’ (e.g. 0.05-0.1 MMes, with 1–3.4M-glycerol and 2–7mM-Mg2+), invariably produces substantial proportions of nonmicrotubule structures such as open sheets, ribbons, and hooked structures. We conclude that the self-assembly of tubulin dimer exclusively into bona fide microtubules occurs over a very restricted range of solution conditions in the normally used Pipes- and Mes-based buffers. Deviation from these conditions readily promotes the formation of mixtures of polymorphic forms. Many buffer systems used for the assembly and disassembly of microtubules composed of tubulin dimer appear likely to promote the formation of structures related to, but significantly different from, normal microtubules. This represents a cautionary factor in the interpretation of in vitro assembly and disassembly properties of microtubules

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The Origin of the Eukaryotic Cell Based on Conservation of Existing Interfaces

Artificial Life ◽

10.1162/artl.2006.12.4.513 ◽

2006 ◽

Vol 12 (4) ◽

pp. 513-523 ◽

Cited By ~ 8

Author(s):

Albert D. G. de Roos

Keyword(s):

Plasma Membrane ◽

Protein Interactions ◽

Self Assembly ◽

Evolutionary Model ◽

Lipid Vesicles ◽

Eukaryotic Cell ◽

The Self ◽

Lipid Protein Interactions

Current theories about the origin of the eukaryotic cell all assume that during evolution a prokaryotic cell acquired a nucleus. Here, it is shown that a scenario in which the nucleus acquired a plasma membrane is inherently less complex because existing interfaces remain intact during evolution. Using this scenario, the evolution to the first eukaryotic cell can be modeled in three steps, based on the self-assembly of cellular membranes by lipid-protein interactions. First, the inclusion of chromosomes in a nuclear membrane is mediated by interactions between laminar proteins and lipid vesicles. Second, the formation of a primitive endoplasmic reticulum, or exomembrane, is induced by the expression of intrinsic membrane proteins. Third, a plasma membrane is formed by fusion of exomembrane vesicles on the cytoskeletal protein scaffold. All three self-assembly processes occur both in vivo and in vitro. This new model provides a gradual Darwinistic evolutionary model of the origins of the eukaryotic cell and suggests an inherent ability of an ancestral, primitive genome to induce its own inclusion in a membrane.

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Yeast proteins associated with microtubules in vitro and in vivo.

Molecular Biology of the Cell ◽

10.1091/mbc.3.1.29 ◽

1992 ◽

Vol 3 (1) ◽

pp. 29-47 ◽

Cited By ~ 57

Author(s):

G Barnes ◽

K A Louie ◽

D Botstein

Keyword(s):

Self Assembly ◽

Synthetic Peptides ◽

Immunofluorescence Microscopy ◽

The Self ◽

Microtubule Associated Proteins ◽

Amino Terminal ◽

Novel Proteins ◽

Associated Proteins

Conditions were established for the self-assembly of milligram amounts of purified Saccharomyces cerevisiae tubulin. Microtubules assembled with pure yeast tubulin were not stabilized by taxol; hybrid microtubules containing substoichiometric amounts of bovine tubulin were stabilized. Yeast microtubule-associated proteins (MAPs) were identified on affinity matrices made from hybrid and all-bovine microtubules. About 25 yeast MAPs were isolated. The amino-terminal sequences of several of these were determined: three were known metabolic enzymes, two were GTP-binding proteins (including the product of the SAR1 gene), and three were novel proteins not found in sequence databases. Affinity-purified antisera were generated against synthetic peptides corresponding to two of the apparently novel proteins (38 and 50 kDa). Immunofluorescence microscopy showed that both these proteins colocalize with intra- and extranuclear microtubules in vivo.

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