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1.
Mol Psychiatry ; 21(11): 1573-1588, 2016 11.
Article in English | MEDLINE | ID: mdl-27698428

ABSTRACT

Human neural progenitors derived from pluripotent stem cells develop into electrophysiologically active neurons at heterogeneous rates, which can confound disease-relevant discoveries in neurology and psychiatry. By combining patch clamping, morphological and transcriptome analysis on single-human neurons in vitro, we defined a continuum of poor to highly functional electrophysiological states of differentiated neurons. The strong correlations between action potentials, synaptic activity, dendritic complexity and gene expression highlight the importance of methods for isolating functionally comparable neurons for in vitro investigations of brain disorders. Although whole-cell electrophysiology is the gold standard for functional evaluation, it often lacks the scalability required for disease modeling studies. Here, we demonstrate a multimodal machine-learning strategy to identify new molecular features that predict the physiological states of single neurons, independently of the time spent in vitro. As further proof of concept, we selected one of the potential neurophysiological biomarkers identified in this study-GDAP1L1-to isolate highly functional live human neurons in vitro.


Subject(s)
Sequence Analysis, RNA/methods , Single-Cell Analysis/methods , Action Potentials/physiology , Cell Differentiation/physiology , Cells, Cultured , Electrophysiology , Humans , Induced Pluripotent Stem Cells/physiology , Machine Learning , Neurons/metabolism , Patch-Clamp Techniques , Pluripotent Stem Cells , RNA
2.
Mol Psychiatry ; 21(1): 49-61, 2016 Jan.
Article in English | MEDLINE | ID: mdl-26503761

ABSTRACT

The brain's serotonergic system centrally regulates several physiological processes and its dysfunction has been implicated in the pathophysiology of several neuropsychiatric disorders. While in the past our understanding of serotonergic neurotransmission has come mainly from mouse models, the development of pluripotent stem cell and induced fibroblast-to-neuron (iN) transdifferentiation technologies has revolutionized our ability to generate human neurons in vitro. Utilizing these techniques and a novel lentiviral reporter for serotonergic neurons, we identified and overexpressed key transcription factors to successfully generate human serotonergic neurons. We found that overexpressing the transcription factors NKX2.2, FEV, GATA2 and LMX1B in combination with ASCL1 and NGN2 directly and efficiently generated serotonergic neurons from human fibroblasts. Induced serotonergic neurons (iSNs) showed increased expression of specific serotonergic genes that are known to be expressed in raphe nuclei. iSNs displayed spontaneous action potentials, released serotonin in vitro and functionally responded to selective serotonin reuptake inhibitors (SSRIs). Here, we demonstrate the efficient generation of functional human serotonergic neurons from human fibroblasts as a novel tool for studying human serotonergic neurotransmission in health and disease.


Subject(s)
Cytological Techniques/methods , Fibroblasts/physiology , Serotonergic Neurons/physiology , Animals , Astrocytes/physiology , Basic Helix-Loop-Helix Transcription Factors/genetics , Basic Helix-Loop-Helix Transcription Factors/metabolism , Cell Line , Cell Transdifferentiation/physiology , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , GATA2 Transcription Factor/genetics , GATA2 Transcription Factor/metabolism , Genetic Vectors , Homeobox Protein Nkx-2.2 , Homeodomain Proteins/genetics , Homeodomain Proteins/metabolism , Human Embryonic Stem Cells/physiology , Humans , LIM-Homeodomain Proteins/genetics , LIM-Homeodomain Proteins/metabolism , Lentivirus/genetics , Mice , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Transcription Factors/genetics , Transcription Factors/metabolism , Transcriptome , Zebrafish Proteins
4.
Biochem Biophys Res Commun ; 266(1): 243-7, 1999 Dec 09.
Article in English | MEDLINE | ID: mdl-10581196

ABSTRACT

Many proteins assemble as oligomeric complexes and in several cases a distinct domain mediates the interaction between the subunits. The identification of new oligomerization modules is relevant to comprehend both the architecture and the evolution of protein sequences and also for protein engineering applications. Using the bacteriophage lambda repressor dimerization assay, we searched Escherichia coli genomic libraries for sequences able to mediate protein oligomerization in vivo. We identified short peptides that can substitute very effectively the dimerizing domain of the repressor. Most of these peptides belong to open reading frames that are normally not expressed in the bacterial cell.


Subject(s)
Bacterial Proteins/metabolism , Bacteriophage lambda/genetics , DNA-Binding Proteins , Escherichia coli/genetics , Peptide Library , Peptides/metabolism , Repressor Proteins/metabolism , Amino Acid Sequence , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Cloning, Molecular , Dimerization , Escherichia coli/chemistry , Escherichia coli/metabolism , Molecular Sequence Data , Open Reading Frames/genetics , Peptides/chemistry , Peptides/genetics , Phenotype , Protein Binding , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Repressor Proteins/chemistry , Repressor Proteins/genetics , Reproducibility of Results , Sequence Deletion , Viral Proteins/genetics , Viral Proteins/metabolism , Viral Regulatory and Accessory Proteins
5.
Biochem Biophys Res Commun ; 250(2): 342-6, 1998 Sep 18.
Article in English | MEDLINE | ID: mdl-9753631

ABSTRACT

Ferritin is an iron-storage protein composed of 24 polypeptide chains which assemble into a hollow shell. Previously, we have shown that a multisubstituted ferritin mutant containing the peptide ESVWNP in place of the wild-type sequence GAPESG in a short exposed loop directs the synthesis of a product that assembles in a conformation remarkably different from that of the normal molecule. We have further characterized this mutant and we have tried to determine which of the substituted residues causes the large conformational change. Reversion of the mutant conformation was obtained changing the three residues WNP back to the wild-type sequence ESG (DE loop: ESVESG). However, the converse three amino acid change GAPWNP produced insoluble and unassembled ferritin. Therefore, the substitutions of GAP by ESV together with ESG by WNP have a largely cooperative and hardly predictable effect.


Subject(s)
Ferritins/chemistry , Protein Conformation , Amino Acid Substitution , Ferritins/genetics , Humans , Mutation
6.
FEBS Lett ; 423(3): 371-5, 1998 Feb 27.
Article in English | MEDLINE | ID: mdl-9515742

ABSTRACT

In order to assess the potentiality of Vibrio cholerae ToxR protein and of bacteriophage lambda repressor as indicators of the dimerization of periplasmic proteins in Escherichia coli, we have constructed a series of plasmids encoding transmembrane fusion proteins. The amino-terminal part, containing the DNA binding domain of either ToxR or lambda repressor, is located in the cytoplasm and acts as reporter for dimerization. As models of periplasmic proteins we have used alkaline phosphatase (a dimer) and beta-lactamase (a monomer). Both the expression level and the distance between the transmembrane segment and the periplasmic protein substantially affect the activity of the reporter domains.


Subject(s)
DNA-Binding Proteins/metabolism , Escherichia coli/chemistry , Gene Expression Regulation, Bacterial/genetics , Periplasm/metabolism , Repressor Proteins/metabolism , Transcription Factors/metabolism , Alkaline Phosphatase/metabolism , Bacterial Proteins/metabolism , DNA-Binding Proteins/genetics , Dimerization , Genes, Reporter/genetics , Membrane Proteins/metabolism , Recombinant Fusion Proteins/metabolism , Repressor Proteins/genetics , Transcription Factors/genetics , Vibrio cholerae/chemistry , Viral Proteins , Viral Regulatory and Accessory Proteins , beta-Lactamases/metabolism
7.
FEBS Lett ; 394(3): 311-5, 1996 Oct 07.
Article in English | MEDLINE | ID: mdl-8830664

ABSTRACT

As a consequence of elevated expression rates, the intracellular aggregation of polypeptide chains is commonly observed in E. coli. Although wild-type human ferritin, a polymeric iron storage protein, accumulates in the soluble form at high level in the bacterial cytoplasmic fraction, some amino acid substitutions in an exposed loop direct the synthesis of a highly insoluble product. We found that two mechanisms can lead to the aggregation of ferritin. While some mutations prevent ferritin polymerisation, others cause the precipitation of molecules in the assembled state.


Subject(s)
Ferritins/chemistry , Mutation , Polymers/chemistry , Protein Structure, Secondary , Biotechnology , Chemical Precipitation , Escherichia coli/genetics , Ferritins/genetics , Humans , Inclusion Bodies , Models, Molecular , Recombinant Proteins/chemistry , Solubility
8.
J Mol Biol ; 259(4): 575-8, 1996 Jun 21.
Article in English | MEDLINE | ID: mdl-8683565

ABSTRACT

The lambda phage repressor is currently used as a genetic tool to analyze homodimeric interactions in Escherichia coli. We have applied this system to detect the interaction that takes place within an enzyme-protein inhibitor complex. The sequences encoding the catalytic subunit of the cAMP-dependent protein kinase and the active portion of the natural thermostable protein kinase inhibitor have been fused to the carboxy terminus of the repressor DNA binding domain and introduced into compatible plasmids. Co-expression of the two gene fusions in E. coli lead to the formation of heterodimers that confer a high level of protection from lambda phage infection. The level of lambda immunity depends specifically upon the amino acid sequence of the interacting proteins, as a single amino acid substitution in the inhibitor peptide (Phe10-Ala) restores the sensitivity phenotype.


Subject(s)
Carrier Proteins/metabolism , Cyclic AMP-Dependent Protein Kinases/metabolism , DNA-Binding Proteins , Intracellular Signaling Peptides and Proteins , Repressor Proteins/genetics , Amino Acid Sequence , Bacteriophage lambda/genetics , Bacteriophage lambda/physiology , Base Sequence , Carrier Proteins/chemistry , Carrier Proteins/genetics , Cloning, Molecular , Cyclic AMP-Dependent Protein Kinases/antagonists & inhibitors , Cyclic AMP-Dependent Protein Kinases/chemistry , Cyclic AMP-Dependent Protein Kinases/genetics , Escherichia coli/physiology , Escherichia coli/virology , Genetic Vectors , Molecular Sequence Data , Mutagenesis, Insertional , Phenotype , Plasmids , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Repressor Proteins/metabolism , Transformation, Bacterial , Viral Proteins , Viral Regulatory and Accessory Proteins
9.
J Mol Biol ; 227(2): 532-43, 1992 Sep 20.
Article in English | MEDLINE | ID: mdl-1404367

ABSTRACT

Although some protein folding theories sustain that the peptides (loops) that connect elements of more compact secondary structure may be important in the folding process, most of the data accumulated until now seems to contradict this notion. To approach this problem we have isolated and characterized a number of mutants in which the amino acid sequence of the peptide that connects helix D and helix E in the H-chain of human ferritin has been randomized. Our results indicate that, though no single loop residue is absolutely required for ferritin to attain the native conformation, most of the mutants that we have obtained by random regional mutagenesis, affect its folding/assembly process. This conclusion was reached utilizing a sensitive test that associates the color formed by a colony synthesizing a hybrid ferritin-beta-galactosidase protein to the ability of the ferritin domain to fold and assemble as the native protein. The characterization of the folding/assembly properties of our collection of mutants and the comparison of the mutant loop sequences, have allowed us to draw the following conclusions. Mutants that have positively charged residues at position 159, 160 or 161 fail to assemble into the native protein shell and form an insoluble aggregate. Interestingly some loop amino acid sequences cause the E-helix to reverse direction and to expose its COOH group, normally hidden inside the protein cavity, to the solvent. The propensity of a given ferritin mutant to fold into this "non-native" conformation can be attenuated by the introduction of Gly at position 159 and 164, as in the natural ferritin.


Subject(s)
Ferritins/chemistry , Ferritins/genetics , Mutation , Protein Folding , Amino Acid Sequence , DNA , Molecular Sequence Data , Phenotype , Proline/chemistry , Solubility
10.
J Biol Chem ; 267(20): 14077-83, 1992 Jul 15.
Article in English | MEDLINE | ID: mdl-1629207

ABSTRACT

Human ferritin, a multimeric iron storage protein, is composed by various proportions of two subunit types: the H- and L-chains. The biological functions of these two genic products have not been clarified, although differences in reactivity with iron have been shown. Starting from the hypothesis that the high stability typical of ferritin is an important property which may be relevant for its iron storage function, we studied ferritin homopolymers of H- and L-chains in different denaturing conditions. In addition we analyzed 13 H-chain variants with alterations in regions conserved within mammalian H-chains. In all the denaturation experiments H-chain ferritin showed lower stability than L-chain ferritin. The difference was greater in guanidine HCl denaturation experiments, where the end products are fully unfolded peptides, than in acidic denaturation experiments, where the end products are peptides with properties analogous to "molten globule." The study on H-chain variants showed: (i) ferritin stability was not affected by alterations of regions exposed to the inner or outer surface of the shell and not involved in intra- or inter-chain interactions; (ii) stability was reduced by alterations of sequences involved in inter-subunit interactions such as the deletion of the N-terminal extension or substitutions along the hydrophobic and hydrophilic channels; (iii) stability was increased by the substitution of 2 amino acids inside the four-helix bundle with those of the homologous L-chain. One of the residues is involved in a salt bridge in the L-chain, and we concluded that the stability difference between H- and L-ferritins is to a large extent due to the stabilizing effect of this salt bridge on the L-subunit fold.


Subject(s)
Ferritins/chemistry , Amino Acid Sequence , Binding Sites , Circular Dichroism , Computer Graphics , Computer Simulation , Escherichia coli/genetics , Ferritins/genetics , Ferritins/isolation & purification , Genetic Variation , Humans , Kinetics , Macromolecular Substances , Models, Molecular , Molecular Sequence Data , Protein Conformation , Protein Denaturation , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification
11.
Nucleic Acids Res ; 20(8): 1859-63, 1992 Apr 25.
Article in English | MEDLINE | ID: mdl-1579486

ABSTRACT

We have analyzed several randomly selected mRNAs, of the relatively abundant category, on the basis of maternal or zygotic origin and translational efficiency at different developmental stages. For this purpose, clones from a Xenopus embryo cDNA library were hybridized with cDNA probes prepared with poly(A)+RNA from polysomes and from mRNPs of embryos at different stages. The results obtained indicate that the majority of the relatively abundant mRNAs (38 out of 61) is subject to some kind of translational regulation during embryogenesis. Moreover, 30 clones have been selected as corresponding to mRNAs that behave, from the point of view of transcriptional and translational regulation, similarly to previously studied ribosomal protein (r-protein) mRNAs. Sequence analysis of 20 of these selected cDNAs has shown that half of them are in fact homologous to already sequenced r-protein mRNAs. Unexpectedly we have found that also the mRNA for alpha-cardiac actin and another mRNA homologous to creatine kinase M mRNA have a similar translational regulation during embryogenesis.


Subject(s)
Embryo, Nonmammalian/metabolism , Gene Expression Regulation/genetics , Protein Biosynthesis/genetics , RNA, Messenger/genetics , Ribosomal Proteins/genetics , Actins/genetics , Amino Acid Sequence , Animals , Base Sequence , Blotting, Northern , Cloning, Molecular , Creatine Kinase/genetics , DNA Probes/genetics , Molecular Sequence Data , Xenopus laevis
12.
J Mol Biol ; 222(2): 301-10, 1991 Nov 20.
Article in English | MEDLINE | ID: mdl-1720463

ABSTRACT

Practically any oligopeptide can be exposed on the surface of the bacteriophage capsid by fusion to the major coat protein of filamentous bacteriophages. A phage expressing a particular peptide tag can be selected from a mixture of tens of millions of clones, exposing oligopeptides of random sequence, by affinity purification with a protein ligand. In this respect, pVIII can be used as an alternative and complement to the exposition vectors based on the product of gene III (pIII). We have constructed a phagemid vector that contains gene VIII under the control of the pLac promoter. This vector can be conveniently used to construct libraries of oligopeptides with a random amino acid sequence. An antipeptide monoclonal antibody was used to affinity-purify phagemids exposing oligopeptides which can interact with the monoclonal antibody. DNA sequencing of the amino terminus of gene VIII of the recovered clones predicts the synthesis of hybrid proteins whose aminoterminal amino acid sequence is related to that of the oligopeptide used to raise the antibody. In other words, only oligopeptides that bind a very small portion of the immunoglobulin G surface are affinity-purified by this method, implying that the antigen binding site possesses molecular properties that renders it much stickier than the remainder of the molecule.


Subject(s)
Antibodies/metabolism , Coliphages/genetics , Epitopes , Oligopeptides/immunology , Amino Acid Sequence , Base Sequence , Capsid/genetics , Gene Library , Genetic Vectors , Ligands , Molecular Sequence Data , Oligopeptides/chemistry , Plasmids , Recombinant Proteins/immunology , Restriction Mapping
13.
Microbiologica ; 7(3): 243-50, 1984 Jul.
Article in English | MEDLINE | ID: mdl-6503731

ABSTRACT

Broth cultures infected with ureaplasmas for 48 hrs were unable to support further growth of reinoculated ureaplasmas even when reconstituted with fresh ureaplasma medium. The apparent toxicity of spent cultures was ascribed to the presence of non viable ureaplasmas still containing a fully active urease since all the procedures adopted to abolish or reduce the urease activity restored the ability of these spent cultures to support ureaplasma growth. However, if the cause of the steep decline phase of ureaplasma cells could be reasonably ascribed to the enzymatic activity of urease present in the dead cells, the reasons for the poor field and the low titers achieved by the organisms during the logarithmic phase of growth remain an enigma.


Subject(s)
Ureaplasma/growth & development , Urease/pharmacology , Ammonium Chloride/pharmacology , Culture Media , Filtration , Hydrogen-Ion Concentration , Urea/pharmacology , Ureaplasma/enzymology , Urease/analysis
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