Artificial cells drive neural differentiation

Toparlak, Ö. Duhan; Zasso, Jacopo; Bridi, Simone; Serra, Mauro Dalla; Macchi, Paolo; Conti, Luciano; Baudet, Marie-Laure; Mansy, Sheref S.

doi:10.1126/sciadv.abb4920

Cited by 86 publications

(103 citation statements)

References 67 publications

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“…[ 18–20 ] More recently, artificial cells were shown to drive the differentiation of neural stem cells. [ 21 ] If similar systems were capable of sensing and synthesizing dopamine, for example, then artificial cells could potentially be used to treat debilitating ailments, such as Parkinson's disease and anxiety. Such therapies would be advantageous, since the needed signaling molecule, in this case dopamine, would be furnished only when needed and would be synthesized from readily available resources, i.e., amino acids.…”

Section: Figurementioning

confidence: 99%

Cell‐Free Synthesis of Dopamine and Serotonin in Two Steps with Purified Enzymes

et al. 2020

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For example, the intermediates of the synthesis of dopamine and serotonin are L-DOPA (L-dihydroxy-phenylalanine) and 5-HTP (5-hydroxytryptophan), respectively, and have found use as administered precursors that are capable of crossing the blood-brain barrier. Although several studies have focused on the characterization of the separate enzymes involved in the biosynthesis of serotonin and dopamine, [3-5] surprisingly little effort has been put into developing enzymatic methods for the production of the final neurotransmitters starting from their proteinogenic amino acids precursors, i.e., L-tryptophan (Trp) and L-tyrosine (Tyr). If developed, such a system may open up new opportunities to build nanofactories and artificial cells for the treatment of neurological disorders. [6-8] To produce the monoamine neurotransmitters serotonin and dopamine in vitro, hydroxylases specific to the amino acid substrate were exploited. The catalytic domain of human tryptophan hydroxylase isoform 2 (TPH) was chosen for the synthesis of 5-HTP from Trp, [9] because this enzyme was previously shown to not be inhibited by high concentrations of the substrate. [10] Two versions of the catalytic domain of rat tyrosine hydroxylase were tested for the production of L-DOPA, including a recombinant, wild type version (rTH) and a truncated construct (ΔTH) that was not inhibited by substrate. [11] Additionally, a hydroxylase from the bacterium Chlamydia pneumoniae, Cpn1046, was tested. Cpn1046 has broader substrate specificity compared to TH and is homologous to eukaryotic aromatic amino acid hydroxylases. [12] Since the aromatic amino acid decarboxylase from Drosophila melanogaster (AADC) is active on both 5-HTP and L-DOPA, [13] a single enzyme was used for the decarboxylation step for the synthesis of both serotonin and dopamine. We found that TPH and ΔTH completely hydroxylated Trp and Tyr to produce the intermediates 5-HTP and L-DOPA, respectively. When TPH and ΔTH were coupled with AADC, serotonin and dopamine were efficiently produced in vitro. Each enzyme (TPH, rTH, ΔΤΗ, AADC, and Cpn1046) expressed well when fused to maltose binding protein (MBP), and each protein was purified in a single step with amylose resin (Figure S1, Supporting Information). However, multiple bands were observed on a SDS-PAGE of Cpn1046. Typically, 50 mg of purified protein was obtained per liter of bacterial culture expressing each construct. Conversely, the use of The synthesis of serotonin and dopamine with purified enzymes is described. Both pathways start from an amino acid substrate and synthesize the monoamine neurotransmitter in two enzymatic steps. The enzymes human tryptophan hydroxylase isoform 2, Rattus norvegicus tyrosine hydroxylase, Chlamydia pneumoniae Cpn1046, and aromatic amino acid decarboxylase from Drosophila melanogaster are recombinantly expressed, purified, and shown to be functional in vitro. The hydroxylases efficiently convert L-DOPA (L-dihydroxy-phenylalanine) and 5-HTP (5-hydroxytryptophan) from L-tyrosine and L-tryptophan, respectiv...

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Section: Figurementioning

confidence: 99%

Cell‐Free Synthesis of Dopamine and Serotonin in Two Steps with Purified Enzymes

et al. 2020

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“…Cells can not only sense the presence of a range of signals at the interface of plasma membranes as involved in extracellular communications but also adapt themselves in terms of morphology and behaviors (31). These signals include physical stimuli (e.g., light and temperature) and the concentrations of chemical compounds (e.g., H + and OH − ) and biomacromolecules (e.g., nucleic acids and proteins) (32)(33)(34).…”

Section: Introductionmentioning

confidence: 99%

Membrane-confined liquid-liquid phase separation toward artificial organelles

et al. 2021

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As the basic unit of life, cells are compartmentalized microreactors with molecularly crowded microenvironments. The quest to understand the cell origin inspires the design of synthetic analogs to mimic their functionality and structural complexity. In this work, we integrate membraneless coacervate microdroplets, a prototype of artificial organelles, into a proteinosome to build hierarchical protocells that may serve as a more realistic model of cellular organization. The protocell subcompartments can sense extracellular signals, take actions in response to these stimuli, and adapt their physicochemical behaviors. The tiered protocells are also capable of enriching biomolecular reactants within the confined organelles, thereby accelerating enzymatic reactions. The ability of signal processing inside protocells allows us to design the Boolean logic gates (NOR and NAND) using biochemical inputs. Our results highlight possible exploration of protocell-community signaling and render a flexible synthetic platform to study complex metabolic reaction networks and embodied chemical computation.

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“…recently took this supporting role a step further, by influencing the differentiation behavior of mammalian cells. More specifically, lipid‐based synthetic cells were used for the differentiation of mouse embryonic stem cell‐derived neural stem cells (mNS) [46] . Interestingly, the authors used a quorum signaling pathway to establish communication between the synthetic cells and mammalian cells (Figure 8A) [24,10] .…”

Section: Synthetic Cells In Biomedical Applicationsmentioning

confidence: 99%

Functional Interactions Between Bottom‐Up Synthetic Cells and Living Matter for Biomedical Applications

2021

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Bottom‐up synthetic cells, where diverse non‐living materials are combined in creative ways in order to construct increasingly life‐like and adaptive systems, are fast approaching a level of function that will enable significant advances in solving specific biomedical challenges. Over the last 10 years, we have seen a wide variety of synthetic cell based approaches to challenges in regulating antimicrobial activity, delivering cargo to mammalian cells, and “growth support”. Despite this progress, there has not been a widespread uptake of synthetic cell technologies in biomedical engineering. In this Review, we highlight both the strengths and limitations of these existing synthetic cell applications, as well as give an overview of the state‐of‐the‐art of synthetic cell technology that has yet been applied to cellular contexts. In doing so we aim to identify opportunities for the advancement of this unique intersection of research fields.

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Artificial cells drive neural differentiation

Cited by 86 publications

References 67 publications

Cell‐Free Synthesis of Dopamine and Serotonin in Two Steps with Purified Enzymes

Cell‐Free Synthesis of Dopamine and Serotonin in Two Steps with Purified Enzymes

Membrane-confined liquid-liquid phase separation toward artificial organelles

Functional Interactions Between Bottom‐Up Synthetic Cells and Living Matter for Biomedical Applications

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