scholarly journals Detection of functional protein domains by unbiased genome-wide forward genetic screening

2017 ◽  
Author(s):  
Mareike Herzog ◽  
Fabio Puddu ◽  
Julia Coates ◽  
Nicola Geisler ◽  
Josep V Forment ◽  
...  
Keyword(s):  
Drug Targets ◽  
Large Scale ◽  
Molecular Mechanisms ◽  
Gene Knockout ◽  
Mammalian Cell Culture ◽  
Three Dimensional ◽  
Embryonic Stem ◽  
Protein Domains ◽  
Functional Protein ◽  
Suppressor Mutations

ABSTRACTGenetic and chemo-genetic interactions have played key roles in elucidating the molecular mechanisms by which certain chemicals perturb cellular functions. Many studies have employed gene knockout collections or gene disruption/depletion strategies to identify routes for evolving resistance to chemical agents. By contrast, searching for point-mutational genetic suppressors that can identify separation- or gain-of-function mutations, has been limited even in simpler, genetically amenable organisms such as yeast, and has not until recently been possible in mammalian cell culture systems. Here, by demonstrating its utility in identifying suppressors of cellular sensitivity to the drugs camptothecin or olaparib, we describe an approach allowing systematic, large-scale detection of spontaneous or chemically-induced suppressor mutations in yeast and in haploid mouse embryonic stem cells in a short timeframe, and with potential applications in essentially any other haploid system. In addition to its utility for molecular biology research, this protocol can be used to identify drug targets and to predict mechanisms leading to drug resistance. Mapping suppressor mutations on the primary sequence or three-dimensional structures of protein suppressor hits provides insights into functionally relevant protein domains, advancing our molecular understanding of protein functions, and potentially helping to improve drug design and applicability.

Three-dimensional cell cultures: from molecular mechanisms to clinical applications

1997 ◽  
Vol 273 (4) ◽  
pp. C1109-C1123 ◽  
Author(s):  
Wolfgang Mueller-Klieser
Keyword(s):  
Molecular Mechanisms ◽  
Three Dimensional ◽  
Embryonic Stem ◽  
Basic Research ◽  
Embryoid Bodies ◽  
Liver Support ◽  
Culture Systems ◽  
Bioartificial Liver Support ◽  
D Cell

This article reviews actual advances in the development and application of three-dimensional (3-D) cell culture systems. Recent therapeutically oriented studies include characterization of multicellular-mediated drug resistance, novel ways of quantifying hypoxia, and new approaches to more efficient immunotherapy. Recent progress toward understanding the development of necrosis in tumor spheroids has been made using novel spheroid models. 3-D cultures have been used for studies on molecular mechanisms involved in invasion and metastasis, with a major focus on the role of E-cadherin. Similarly, tumor angiogenesis and the significance of vascular endothelial growth factor have been investigated in a variety of 3-D culture systems. There are many ongoing developments in tissue modeling or remodeling that promise significant progress toward the development of bioartificial liver support and artificial blood. Perhaps one of the most interesting areas of basic research with 3-D cultures is the characterization of embryoid bodies obtained from stable embryonic stem cells. These models have greatly increased the understanding of embryonic development, in particular through the notable exceptional advances in cardiogenesis.

scholarly journals Understanding the yeast host cell response to recombinant membrane protein production

2011 ◽  
Vol 39 (3) ◽  
pp. 719-723 ◽  
Author(s):  
Zharain Bawa ◽  
Charlotte E. Bland ◽  
Nicklas Bonander ◽  
Nagamani Bora ◽  
Stephanie P. Cartwright ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Drug Targets ◽  
Large Scale ◽  
Protein Production ◽  
Molecular Mechanisms ◽  
New Drugs ◽  
Host Cells ◽  
Host Cell Response ◽  
Wide Range

Membrane proteins are drug targets for a wide range of diseases. Having access to appropriate samples for further research underpins the pharmaceutical industry's strategy for developing new drugs. This is typically achieved by synthesizing a protein of interest in host cells that can be cultured on a large scale, allowing the isolation of the pure protein in quantities much higher than those found in the protein's native source. Yeast is a popular host as it is a eukaryote with similar synthetic machinery to that of the native human source cells of many proteins of interest, while also being quick, easy and cheap to grow and process. Even in these cells, the production of human membrane proteins can be plagued by low functional yields; we wish to understand why. We have identified molecular mechanisms and culture parameters underpinning high yields and have consolidated our findings to engineer improved yeast host strains. By relieving the bottlenecks to recombinant membrane protein production in yeast, we aim to contribute to the drug discovery pipeline, while providing insight into translational processes.

scholarly journals Unraveling mitotic protein networks by 3D multiplexed epitope drug screening

2017 ◽  
Author(s):  
Lorenz Maier ◽  
Stefan Kallenberger ◽  
Katharina Jechow ◽  
Marcel Waschow ◽  
Roland Eils ◽  
...  
Keyword(s):  
Drug Targets ◽  
Network Effects ◽  
Protein Localization ◽  
Three Dimensional ◽  
Immunofluorescent Staining ◽  
Functional Protein ◽  
Spatially Resolved ◽  
Antibody Staining ◽  
Drug Assays ◽  
Automated Technology

Three-dimensional protein localization intricately determines the functional coordination of cellular processes. The complex spatial context of protein landscape has been assessed by multiplexed immunofluorescent staining1–3or mass spectrometry4, applied to 2D cell culture with limited physiological relevance5or tissue sections. Here, we present3D SPECS, an automated technology for3DSpatial characterization ofProteinExpressionChanges by microscopic Screening. This workflow encompasses iterative antibody staining of proteins, high-content imaging, and machine learning based classification of mitotic states. This is followed by mapping of spatial protein localization into a spherical, cellular coordinate system, the basis used for model-based prediction of spatially resolved affinities of various mitotic proteins. As a proof-of-concept, we mapped twelve epitopes in 3D cultured epithelial breast spheroids and investigated the network effects of mitotic cancer drugs with known limited success in clinical trials6–8. Our approach reveals novel insights into spindle fragility and global chromatin stress, and predicts unknown interactions between proteins in specific mitotic pathways.3D SPECS’sability to map potential drug targets by multiplexed immunofluorescence in 3D cell cultured models combined with our automized high content assay will inspire future functional protein expression and drug assays.

scholarly journals Cellular-resolution gene expression profiling in the neonatal marmoset brain reveals dynamic species- and region-specific differences

2021 ◽  
Vol 118 (18) ◽  
pp. e2020125118
Author(s):  
Yoshiaki Kita ◽  
Hirozumi Nishibe ◽  
Yan Wang ◽  
Tsutomu Hashikawa ◽  
Satomi S. Kikuchi ◽  
...  
Keyword(s):  
Gene Expression ◽  
Gene Expression Profiling ◽  
Expression Profiling ◽  
Large Scale ◽  
Molecular Mechanisms ◽  
Neural Circuit ◽  
Expression Patterns ◽  
Three Dimensional ◽  
Control Of Gene Expression ◽  
Cellular Resolution

Precise spatiotemporal control of gene expression in the developing brain is critical for neural circuit formation, and comprehensive expression mapping in the developing primate brain is crucial to understand brain function in health and disease. Here, we developed an unbiased, automated, large-scale, cellular-resolution in situ hybridization (ISH)–based gene expression profiling system (GePS) and companion analysis to reveal gene expression patterns in the neonatal New World marmoset cortex, thalamus, and striatum that are distinct from those in mice. Gene-ontology analysis of marmoset-specific genes revealed associations with catalytic activity in the visual cortex and neuropsychiatric disorders in the thalamus. Cortically expressed genes with clear area boundaries were used in a three-dimensional cortical surface mapping algorithm to delineate higher-order cortical areas not evident in two-dimensional ISH data. GePS provides a powerful platform to elucidate the molecular mechanisms underlying primate neurobiology and developmental psychiatric and neurological disorders.

scholarly journals Microscopic Chromosomal Structural and Dynamical Origin of Cell Differentiation and Reprogramming

2020 ◽  
Author(s):  
Xiakun Chu ◽  
Jin Wang
Keyword(s):  
Cell Differentiation ◽  
Large Scale ◽  
Chromosome Structure ◽  
Structural Characteristics ◽  
Three Dimensional ◽  
Embryonic Stem ◽  
Switching Model ◽  
Somatic Cell Reprogramming ◽  
Fundamental Process ◽  
Chromosomal Loci

AbstractAs an essential and fundamental process of life, cell development involves large-scale reorganization of the three-dimensional genome architecture, which forms the basis of gene regulation. Here, we develop a landscape-switching model to explore the microscopic chromosomal structural origin of the embryonic stem cell (ESC) differentiation and the somatic cell reprogramming. We show that chromosome structure exhibits significant compartment-switching in the unit of topologically associating domain. We find that the chromosome during differentiation undergoes monotonic compaction with spatial re-positioning of active and inactive chromosomal loci towards the chromosome surface and interior, respectively. In contrast, an over-expanded chromosome, which exhibits universal localization of loci at the chromosomal surface with erasing the structural characteristics formed in the somatic cells, is observed during reprogramming. We suggest an early distinct differentiation pathway from the ESC to the terminally differentiated cell, giving rise to early bifurcation on the Waddington landscape for the ESC differentiation. Our theoretical model including the non-equilibrium effects, draws a picture of the highly irreversible cell differentiation and reprogramming processes, in line with the experiments. The predictions from our model provide a physical understanding of cell differentiation and reprogramming from the chromosomal structural and dynamical perspective and can be tested by future experiments.

scholarly journals Screening Genes Promoting Exit from Naive Pluripotency Based on Genome-Scale CRISPR-Cas9 Knockout

2020 ◽  
Vol 2020 ◽  
pp. 1-15 ◽  
Author(s):  
Bin Yang ◽  
Junqi Kuang ◽  
Chuman Wu ◽  
Wenyi Zhou ◽  
Shuoji Zhu ◽  
...  
Keyword(s):  
Stem Cell ◽  
Regenerative Medicine ◽  
Large Scale ◽  
Gene Knockout ◽  
Embryonic Stem ◽  
Specific Cell ◽  
Medicine Research ◽  
Model Based ◽  
Key Genes ◽  
Genome Scale

Two of the main problems of stem cell and regenerative medicine are the exit of pluripotency and differentiation to functional cells or tissues. The answer to these two problems holds great value in the clinical translation of stem cell as well as regenerative medicine research. Although piling researches have revealed the truth about pluripotency maintenance, the mechanisms underlying pluripotent cell self-renewal, proliferation, and differentiation into specific cell lineages or tissues are yet to be defined. To this end, we took full advantage of a novel technology, namely, the genome-scale CRISPR-Cas9 knockout (GeCKO). As an effective way of introducing targeted loss-of-function mutations at specific sites in the genome, GeCKO is able to screen in an unbiased manner for key genes that promote exit from pluripotency in mouse embryonic stem cells (mESCs) for the first time. In this study, we successfully established a model based on GeCKO to screen the key genes in pluripotency withdrawal. Our strategies included lentiviral package and infection technology, lenti-Cas9 gene knockout technology, shRNA gene knockdown technology, next-generation sequencing, model-based analysis of genome-scale CRISPR-Cas9 knockout (MAGeCK analysis), GO analysis, and other methods. Our findings provide a novel approach for large-scale screening of genes involved in pluripotency exit and offer an entry point for cell fate regulation research.

A model of the large-scale organization of chromatin

2013 ◽  
Vol 41 (2) ◽  
pp. 508-512 ◽  
Author(s):  
Mariano Barbieri ◽  
Mita Chotalia ◽  
James Fraser ◽  
Liron-Mark Lavitas ◽  
Josée Dostie ◽  
...  
Keyword(s):  
Experimental Data ◽  
Present Article ◽  
Cell Nucleus ◽  
Large Scale ◽  
Spatial Organization ◽  
Molecular Mechanisms ◽  
Three Dimensional ◽  
Polymer Physics ◽  
Self Organization ◽  
Dna Binding Factors

In the cell nucleus, chromosomes have a complex spatial organization, spanning several length scales, which serves vital functional purposes. It is unknown, however, how their three-dimensional architecture is orchestrated. In the present article, we review the application of a model based on classical polymer physics, the strings and binders switch model, to explain the molecular mechanisms of chromatin self-organization. We explore the scenario where chromatin architecture is shaped and regulated by the interactions of chromosomes with diffusing DNA-binding factors via thermodynamics mechanisms and compare it with available experimental data.

scholarly journals Investigating Pathogenetic Mechanisms of Alzheimer’s Disease by Systems Biology Approaches for Drug Discovery

2021 ◽  
Vol 22 (20) ◽  
pp. 11280
Author(s):  
Shan-Ju Yeh ◽  
Ming-Hsun Chung ◽  
Bor-Sen Chen
Keyword(s):  
Alzheimer’S Disease ◽  
Alzheimer's Disease ◽  
Drug Targets ◽  
Large Scale ◽  
Molecular Mechanisms ◽  
Neurodegenerative Disorder ◽  
Early Stage ◽  
Amyloid Β ◽  
Detection Scheme ◽  
Pathogenetic Mechanisms

Alzheimer’s disease (AD) is the most common cause of dementia, characterized by progressive cognitive decline and neurodegenerative disorder. Abnormal aggregations of intracellular neurofibrillary tangles (NFTs) and unusual accumulations of extracellular amyloid-β (Aβ) peptides are two important pathological features in AD brains. However, in spite of large-scale clinical studies and computational simulations, the molecular mechanisms of AD development and progression are still unclear. In this study, we divided all of the samples into two groups: early stage (Braak score I-–III) and later stage (Braak score IV–VI). By big database mining, the candidate genetic and epigenetic networks (GEN) have been constructed. In order to find out the real GENs for two stages of AD, we performed systems identification and system order detection scheme to prune false positives with the help of corresponding microarray data. Applying the principal network projection (PNP) method, core GENs were extracted from real GENs based on the projection values. By the annotation of KEGG pathway, we could obtain core pathways from core GENs and investigate pathogenetic mechanisms for the early and later stage of AD, respectively. Consequently, according to pathogenetic mechanisms, several potential biomarkers are identified as drug targets for multiple-molecule drug design in the treatment of AD.

scholarly journals Functional Dependency Analysis Identifies Potential Druggable Targets in Acute Myeloid Leukemia

Cancers ◽  
2020 ◽  
Vol 12 (12) ◽  
pp. 3710
Author(s):  
Yujia Zhou ◽  
Gregory P. Takacs ◽  
Jatinder K. Lamba ◽  
Christopher Vulpe ◽  
Christopher R. Cogle
Keyword(s):  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Drug Targets ◽  
Gene Knockout ◽  
Three Dimensional ◽  
Term Survival ◽  
Genome Wide ◽  
Druggable Targets ◽  
Acute Myeloid

Refractory disease is a major challenge in treating patients with acute myeloid leukemia (AML). Whereas the armamentarium has expanded in the past few years for treating AML, long-term survival outcomes have yet to be proven. To further expand the arsenal for treating AML, we searched for druggable gene targets in AML by analyzing screening data from a lentiviral-based genome-wide pooled CRISPR-Cas9 library and gene knockout (KO) dependency scores in 15 AML cell lines (HEL, MV411, OCIAML2, THP1, NOMO1, EOL1, KASUMI1, NB4, OCIAML3, MOLM13, TF1, U937, F36P, AML193, P31FUJ). Ninety-four gene KOs met the criteria of (A) specifically essential to AML cell survival, (B) non-essential in non-AML cells, and (C) druggable according to three-dimensional (3D) modeling or ligand-based druggability scoring. Forty-four of 94 gene-KOs (47%) had an already-approved drug match and comprised a drug development list termed “deKO.” Fifty of 94 gene-KOs (53%) had no drug in development and comprised a drug discovery list termed “disKO.” STRING analysis and gene ontology categorization of the disKO targets preferentially cluster in the metabolic processes of UMP biosynthesis, IMP biosynthesis, dihydrofolate metabolism, pyrimidine nucleobase biosynthesis, vitellogenesis, and regulation of T cell differentiation and hematopoiesis. Results from this study serve as a testable compendium of AML drug targets that, after validation, may be translated into new therapeutics.

scholarly journals G9a/GLP-Sensitivity of H3K9me2 Demarcates Two Types of Genomic Compartments

2020 ◽  
Author(s):  
Zixiang Yan ◽  
Luzhang Ji ◽  
Xiangru Huo ◽  
Qianfeng Wang ◽  
Yuwen Zhang ◽  
...  
Keyword(s):  
Genome Organization ◽  
Molecular Mechanisms ◽  
Three Dimensional ◽  
Embryonic Stem ◽  
Nuclear Lamina ◽  
Hierarchical Architecture ◽  
Functional Roles ◽  
3D Genome ◽  
Differentiated Cells ◽  
Mouse Hepatocytes

AbstractIn the nucleus, chromatin is folded into hierarchical architecture that is tightly linked to various nuclear functions. However, the underlying molecular mechanisms that confer these architectures remain incompletely understood. Here, we investigated the functional roles of H3 lysine 9 dimethylation (H3K9me2), one of the abundant histone modifications, in three-dimensional (3D) genome organization. Unlike mouse embryonic stem cells (mESCs), inhibition of methyltransferases G9a and GLP in differentiated cells eliminated H3K9me2 predominantly at A-type (active) genomic compartments, and the level of residual H3K9me2 modification was strongly associated with genomic compartments in differentiated cells. Furthermore, chemical inhibition of G9a/GLP in mouse hepatocytes led to the decreased chromatin-nuclear lamina interactions mainly at G9a/GLP sensitive regions (GSRs), the increased degree of genomic compartmentalization, and the up-regulation of hundreds of genes that were associated with alterations of the 3D chromatin. Collectively, our data demonstrated essential roles of H3K9me2 in 3D genome organization.

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