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Ribosome Mediated Quinary Interactions Modulate In-Cell Protein Activities.

ABSTRACT: Ribosomes are present inside bacterial cells at micromolar concentrations and occupy up to 20% of the cell volume. Under these conditions, even weak quinary interactions between ribosomes and cytosolic proteins can affect protein activity. By using in-cell and in vitro NMR spectroscopy, and biophysical techniques, we show that the enzymes, adenylate kinase and dihydrofolate reductase, and the respective coenzymes, ATP and NADPH, bind to ribosomes with micromolar affinity, and that this interaction suppresses the enzymatic activities of both enzymes. Conversely, thymidylate synthase, which works together with dihydrofolate reductase in the thymidylate synthetic pathway, is activated by ribosomes. We also show that ribosomes impede diffusion of green fluorescent protein in vitro and contribute to the decrease in diffusion in vivo. These results strongly suggest that ribosome-mediated quinary interactions contribute to the differences between in vitro and in vivo protein activities and that ribosomes play a previously under-appreciated nontranslational role in regulating cellular biochemistry.

SUBMITTER: DeMott CM

PROVIDER: S-EPMC5956540 | biostudies-literature | 2017 Aug

REPOSITORIES: biostudies-literature

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Ribosome Mediated Quinary Interactions Modulate In-Cell Protein Activities.

DeMott Christopher M CM Majumder Subhabrata S Burz David S DS Reverdatto Sergey S Shekhtman Alexander A

Biochemistry 20170803 32

Ribosomes are present inside bacterial cells at micromolar concentrations and occupy up to 20% of the cell volume. Under these conditions, even weak quinary interactions between ribosomes and cytosolic proteins can affect protein activity. By using in-cell and in vitro NMR spectroscopy, and biophysical techniques, we show that the enzymes, adenylate kinase and dihydrofolate reductase, and the respective coenzymes, ATP and NADPH, bind to ribosomes with micromolar affinity, and that this interacti ...[more]

PMID: 28715177

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Project description:The functional maturation status of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) has a notable impact upon their use in pharmacological studies, disease modeling, and therapeutic applications. Non-cardiomyocytes (non-CMs) produced in the differentiation process have previously been identified as having an extrinsic influence upon hiPSC-CM development, yet the underlying mechanisms are not fully understood. Herein, we aimed to modulate electrophysiological properties of hiPSC-CMs within co-cultures containing varied proportions of non-CMs and investigate the nature of interactions between these different cell types. Therefore, we sorted cardiac differentiations on day 10 and subsequently replated the cells at ratios of 7:3, 1:1, 3:7, and 1:9 non-CMs to CMs. After a month of co-culture, we evaluated electrophysiological properties through the genetically encoded voltage indicator ArcLight. We ultimately identified that co-cultures with approximately 70%-90% CM purity demonstrated the highest action potential (AP) amplitude and maximum upstroke velocity by day 40 of differentiation, indicative of enhanced electrophysiological maturation, as well as more ventricular-like AP morphologies. Notably, these findings were distinct from those observed for co-cultures of hiPSC-CMs and dermal fibroblasts. We determined that the co-culture phenotypes could not be attributed to paracrine effects of non-CMs due to the inability of conditioned media to recapitulate the observed effects. This led to the further observation of a distinctive expression pattern of connexin 43 (Cx43) at cell-cell interfaces between both CMs and non-CMs. Depletion of Cx43 by short hairpin RNA (shRNA) specifically in the non-CM population within a co-culture environment was able to recapitulate electrophysiological phenotypes of a purer hiPSC-CM population. Collectively, our data demonstrate that abundant non-CM content exerts a significant negative influence upon the electrophysiological maturation of hiPSC-CMs through Cx43-mediated cell-cell-contacts, and thus should be considered regarding the future production of purpose-built hiPSC-CM systems.

Dataset Information

Ribosome Mediated Quinary Interactions Modulate In-Cell Protein Activities.

Publications

Ribosome Mediated Quinary Interactions Modulate In-Cell Protein Activities.

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