Studies on PAF1 regulation and function of recombinant RIG-I

DoctorProteins in intracellular and extracellular regions are crucial players for most biological processes. In the extracellular matrix, proteins can work as messengers or sensors for environmental changes and trigger proper responses in cells. Intracellular proteins accomplish the appropriate reaction to environmental changes and maintain cellular homeostasis on their own or as members of protein complexes. Transcription is a sophisticated biochemical reaction accomplished by diverse proteins and protein complexes to respond to environmental changes or maintain homeostasis. Among the protein complexes, the PAF complex (PAFc), a RNA polymerase II-associated factor complex, participates in transcription, from initiation to termination, by recruiting proteins to proper loci. PAFc is an evolutionarily conserved, multi-subunit protein complex composed of PAF1, CTR9, CDC73, LEO1, and RTF1. Each component of PAFc has individual roles in a specific signaling pathway. Furthermore, the individual components are crucial for the stability of other proteins and complex formation; however, the mechanism of regulation of each component remains undiscovered. Based on that, I studied on the regulation of PAF1, a pivotal scaffold protein of PAFc. PAF1 protein is controlled by CNOT4, one protein of the CCR4-NOT4 complex. CNOT4 regulates PAF1 specifically, not the other members of PAFc, at the protein step. CNOT4 triggers the K48 poly-ubiquitination of PAF1 and 26S proteasome-dependent degradation. Furthermore, CNOT4 or 26S proteasome-dependent degradation of PAF1 protein is required for nuclear localization; however, chromatin binding of PAF1 is not needed. CNOT4 controls chromatin-unbound PAF1 degradation through the 26S proteasome by triggering poly-ubiquitination. Inflammation is orchestrated by intracellular and extracellular proteins and controlled by various signals. Although many inflammation mediators are newly synthesized and released outside of the cell, some are just secreted. The secretion of intracellular proteins is modulated by caspase-1, one protein of the inflammasome complex. Activated caspase-1 triggers the release of IL-1α, IL-1β, MIF, and HMGB1. These proteins function as mediators of inflammation. RIG-I is a cytosolic receptor for viral nucleic acid and induces the antiviral response. RIG-I is also released to the extracellular compartment when caspase-1 is activated by LPS or Poly I:C. Because caspase-1-mediated secreted proteins can control inflammation outside of the cell, I examined the role of RIG-I in the extracellular matrix. To study the role of extracellular RIG-I, I purified recombinant RIG-I and treated it to cells. Although intracellular RIG-I can control the antiviral response, extracellular RIG-I (exRIG-I) cannot affect it. Unexpectedly, exRIG-I can induce mitochondrial enzyme activity and the expression of mitochondrial biogenesis-related transcription regulators and mitochondrial biogenesis. Among the induced genes, PGC1α works as the intracellular mediator of exRIG-I. ExRIG-I increases intracellular ATP and decreases the intracellular lipid. Accumulated lipid during inflammation, a condition for RIG-I secretion, is also decreased by exRIG-I. Furthermore, amino acids 217-925 of RIG-I are necessary for exRIG-I’s effects on mitochondria. Taken together, I suggest that exRIG-I can modulate mitochondrial biogenesis and its function

Recombinant human Retinoic Acid Inducible Gene-I proteins modulates the mitochondrial activity

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Protein stability control of Paf1 by CCR4-NOT complex

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The regulation of PAF1 protein, one of the PAFc, by CCR4-NOT complex

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