Reprogramming cellular functions with engineered membrane proteins

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Switching control of an essential gene for reprogramming of cellular phenotypes in Escherichia coli

Biological systems designs require various dynamic controllers capable of modulating cellular phenotypes to adapt to changing environments. Cellular phenotypes are simultaneously affected by combinations of multiple genes that are controlled by global regulators. However, it is difficult to intentionally control the expression of these global regulators dynamically because they are essential for cell survival and are involved in regulatory networks clustered in operons. Here, we designed a platform that allows dynamic modulation of the expression of an essential gene. Using this system, comprising of on/off switches that respond to an extracellular stimulus, we successfully demonstrated the switching control of the expression of fusA encoding elongation factor G (EF-G). An additional control module in this system that responds to changed external signals was shown to provide the capacity to "switch gears" and reprogram cellular phenotypes with desired timing.     

Engineering cytokine receptors to control cellular functions

Cytokine receptors function as an interface between a cell and the extracellular milieu, and play a pivotal role in cell fate, because they recognize specific ligands via their extracellular domain and trigger signal transduction via their intracellular domain. Recent advances in unraveling the mechanism of cytokine receptor-mediated signal transduction have allowed us to engineer cytokine receptors with distinct functions, which have a potential for use in biotechnology. This paper reviews the history and current topics of receptor engineering.     

Focus on induced pluripotency and cellular reprogramming

Reflecting on the opportunities that ‘induced pluripotency’ offers for basic research and clinical translation, the 2015 Focus of The EMBO Journal highlights some of the most challenging biological questions studied using advanced iPSC‐based technologies.     

Small molecule-based reversible reprogramming of cellular lifespan

Most somatic cells encounter an inevitable destiny, senescence. Little progress has been made in identifying small molecules that extend the finite lifespan of normal human cells. Here we show that the intrinsic 'senescence clock' can be reset in a reversible manner by selective modulation of the ataxia telangiectasia-mutated (ATM) protein and ATM- and Rad3-related (ATR) protein with a small molecule, CGK733. This compound was identified by a high-throughput phenotypic screen with automated imaging. Employing a magnetic nanoprobe technology, magnetism-based interaction capture (MAGIC), we identified ATM as the molecular target of CGK733 from a genome-wide screen. CGK733 inhibits ATM and ATR kinase activities and blocks their checkpoint signaling pathways with great selectivity. Consistently, siRNA-mediated knockdown of ATM and ATR induced the proliferation of senescent cells, although with lesser efficiency than CGK733. These results might reflect the specific targeting of the kinase activities of ATM and ATR by CGK733 without affecting any other domains required for cell proliferation.     

The glycerophosphoinositols and their cellular functions

Interest in the glycerophosphoinositols has been increasing recently, on the basis of their biological activities. The cellular metabolism of these water-soluble bioactive phosphoinositide metabolites has been clarified, with the identification of the specific enzyme involved in their synthesis, PLA2IVα (phospholipase A2 IVα), and the definition of their phosphodiesterase-based catabolism, and thus inactivation. The functional roles and mechanisms of action of these compounds have been investigated in different cellular contexts. This has led to their definition in the control of various cell functions, such as cell proliferation in the thyroid and actin cytoskeleton organization in fibroblasts and lymphocytes. Roles for the glycerophosphoinositols in immune and inflammatory responses are also being defined. In addition to these physiological functions, the glycerophosphoinositols have potential anti-metastatic activities that should lead to their pharmacological exploitation.     

The use of zeolite y in the purification of intra cellular accumulated proteins from genetically engineered cells

Summary Fast and efficient release of intracellular proteins by the use of detergents followed by immediate removal of the detergent by adsorption to zeolite Y is described. The removal of the detergent makes it possible to continue the purification process without interference by the detergent and the process is exemplified by the purification of protein A fromE. coli encoded by the plasmid pRIT-2T. Compared to disruption of the cells by sonication, increased stability against proteolytic degradation was obtained.     

GATA family members as inducers for cellular reprogramming to pluripotency

Members of the GATA protein family play important roles in lineage specification and transdifferentiation. Previous reports show that some members of the GATA protein family can also induce pluripotency in somatic cells by substituting for Oct4, a key pluripotency-associated factor. However, the mechanism linking lineage-specifying cues and the activation of pluripotency remains elusive. Here, we report that all GATA family members can substitute for Oct4 to induce pluripotency. We found that all members of the GATA family could inhibit the overrepresented ectodermal-lineage genes, which is consistent with previous reports indicating that a balance of different lineage-specifying forces is important for the restoration of pluripotency. A conserved zinc-finger DNA-binding domain in the C-terminus is critical for the GATA family to induce pluripotency. Using RNA-seq and ChIP-seq, we determined that the pluripotency-related gene Sall4 is a direct target of GATA family members during reprogramming and serves as a bridge linking the lineage-specifying GATA family to the pluripotency circuit. Thus, the GATA family is the first protein family of which all members can function as inducers of the reprogramming process and can substitute for Oct4. Our results suggest that the role of GATA family in reprogramming has been underestimated and that the GATA family may serve as an important mediator of cell fate conversion.     

Probing p53 biological functions through the use of genetically engineered mouse models

The p53 tumor suppressor gene is rendered dysfunctional in the majority of human cancers. To model the effects of p53 dysfunction in an experimentally manipulable organismal context, genetically engineered inbred mice have been the models of choice. Transgenic and knock-out technologies have been utilized to generate an array of different p53 germ line alterations. As expected, many (though not all) of the mutant p53 mouse models are susceptible to enhanced spontaneous and carcinogen-induced tumors of a variety of types. A number of different variables affect the incidence and spectrum of tumors in p53 mutant mice. These include strain background, the nature of the p53 mutation, the presence of wild-type p53 (in addition to mutant p53), exposure to physical and chemical mutagens, or introduction of other cancer-associated genes into the mutant p53 background. In addition to their role in furthering our understanding of the mechanisms of cancer initiation and progression, these models have led to unexpected insights into p53 function in embryogenesis and aging. With the development of ever more sophisticated methods for manipulating the mouse genome, new p53 models are on the horizon, which should deliver advances that will provide not only important mechanistic insights but also discoveries of great clinical relevance.     

Potential of transposon-mediated cellular reprogramming towards cell-based therapies

Induced pluripotent stem(iPS) cells present a seminal discovery in cell biology and promise to support innovative treatments of so far incurable diseases. To translate i PS technology into clinical trials, the safety and stability of these reprogrammed cells needs to be shown. In recent years, different non-viral transposon systems have been developed for the induction of cellular pluripotency, and for the directed differentiation into desired cell types. In this review, we summarize the current state of the art of different transposon systems in i PS-based cell therapies.