Cellular Regulatory Molecule Research Articles

Acetate Revisited: A Key Biomolecule at the Nexus of Metabolism, Epigenetics and Oncogenesis-Part 1: Acetyl-CoA, Acetogenesis and Acyl-CoA Short-Chain Synthetases.

Acetate is a major end product of bacterial fermentation of fiber in the gut. Acetate, whether derived from the diet or from fermentation in the colon, has been implicated in a range of health benefits. Acetate is also generated in and released from various tissues including the intestine and liver, and is generated within all cells by deacetylation reactions. To be utilized, all acetate, regardless of the source, must be converted to acetyl coenzyme A (acetyl-CoA), which is carried out by enzymes known as acyl-CoA short-chain synthetases. Acyl-CoA short-chain synthetase-2 (ACSS2) is present in the cytosol and nuclei of many cell types, whereas ACSS1 is mitochondrial, with greatest expression in heart, skeletal muscle, and brown adipose tissue. In addition to acting to redistribute carbon systemically like a ketone body, acetate is becoming recognized as a cellular regulatory molecule with diverse functions beyond the formation of acetyl-CoA for energy derivation and lipogenesis. Acetate acts, in part, as a metabolic sensor linking nutrient balance and cellular stress responses with gene transcription and the regulation of protein function. ACSS2 is an important task-switching component of this sensory system wherein nutrient deprivation, hypoxia and other stressors shift ACSS2 from a lipogenic role in the cytoplasm to a regulatory role in the cell nucleus. Protein acetylation is a critical post-translational modification involved in regulating cell behavior, and alterations in protein acetylation status have been linked to multiple disease states, including cancer. Improving our fundamental understanding of the “acetylome” and how acetate is generated and utilized at the subcellular level in different cell types will provide much needed insight into normal and neoplastic cellular metabolism and the epigenetic regulation of phenotypic expression under different physiological stressors. This article is Part 1 of 2 – for Part 2 see doi: 10.3389/fphys.2020.580171.

The Role of Long Noncoding RNAs in Human Papillomavirus-associated Pathogenesis.

Infections with high-risk human papillomaviruses cause ~5% of all human cancers. E6 and E7 are the only viral genes that are consistently expressed in cancers, and they are necessary for tumor initiation, progression, and maintenance. E6 and E7 encode small proteins that lack intrinsic enzymatic activities and they function by binding to cellular regulatory molecules, thereby subverting normal cellular homeostasis. Much effort has focused on identifying protein targets of the E6 and E7 proteins, but it has been estimated that ~98% of the human transcriptome does not encode proteins. There is a growing interest in studying noncoding RNAs as biochemical targets and biological mediators of human papillomavirus (HPV) E6/E7 oncogenic activities. This review focuses on HPV E6/E7 targeting cellular long noncoding RNAs, a class of biologically versatile molecules that regulate almost every known biological process and how this may contribute to viral oncogenesis.

Biotin-based Pulldown Assay to Validate mRNA Targets of Cellular miRNAs.

MicroRNAs (miRNAs) are a class of small noncoding RNAs that post-transcriptionally regulate cellular gene expression. MiRNAs bind to the 3' untranslated region (UTR) of target mRNA to inhibit protein translation or in some instances cause mRNA degradation. The binding of the miRNA to the 3' UTR of the target mRNA is mediated by a 2-8 nucleotide seed sequence at the 5' end of miRNA. While the role of miRNAs as cellular regulatory molecules is well established, identification of the target mRNAs with functional relevance remains a challenge. Bioinformatic tools have been employed to predict sequences within the 3' UTR of mRNAs as potential targets for miRNA binding. These tools have also been utilized to determine the evolutionary conservation of such sequences among related species in an attempt to predict functional role. However, these computational methods often generate false positive results and are limited to predicting canonical interaction between miRNA and mRNA. Therefore, experimental procedures that measure direct binding of miRNA to its mRNA target are necessary to establish functional interaction. In this report, we describe a sensitive method for validating direct interaction between the cellular miRNA miR-125b and the 3' UTR of PARP-1 mRNA. We elaborate a protocol in which synthetic biotinylated-miRNA mimics were transfected into mammalian cells and the miRNA-mRNA complex in the cellular lysate was pulled down with streptavidin-coated magnetic beads. Finally, the target mRNA in the pulled-down nucleic acid complex was quantified using a qPCR-based strategy.

Neurotransmitters as early signals for central nervous system development.

During brain ontogenesis, the temporal and spatial generation of the different types of neuronal and glial cells from precursors occurs as a sequence of successive progenitor stages whose proliferation, survival and cell-fate choice are controlled by environmental and cellular regulatory molecules. Neurotransmitters belong to the chemical microenvironment of neural cells, even at the earliest stages of brain development. It is now established that specific neurotransmitter receptors are present on progenitor cells of the developing central nervous system and could play, during neural development, a role that has remained unsuspected until recently. The present review focuses on the occurrence of neurotransmitters and their corresponding ligand-gated ion channel receptors in immature cells, including neural stem cells of specific embryonic and neonatal brain regions. We summarize in vitro and in vivo data arguing that neurotransmitters could regulate morphogenetic events such as proliferation, growth, migration, differentiation and survival of neural precursor cells. The understanding of neurotransmitter function during early neural maturation could lead to the development of pharmacological tools aimed at improving adult brain repair strategies.

Emerging concepts in the Ras superfamily of GTP-binding proteins.

The Ras superfamily of GTP-binding proteins (> 50 members) regulates a diverse spectrum of intracellular processes. These include cellular proliferation and differentiation, intracellular vesicular trafficking, cytoskeletal control, NADPH oxidase function, as well as others. In this review, we describe recent progress and emerging themes in the action and regulation of these important cellular regulatory molecules. Structural studies have provided insight into the function of low molecular weight GTP-binding proteins (LMWGs) as molecular switches, and are defining modes of interaction with associated regulatory molecules. Details of the enzymatic processes involved in the posttranslational processing of LMWGs, and how this processing is important for protein function, are being elucidated. A variety of GTPase activating proteins, GDP/GTP dissociation stimulators, and GDP dissociation inhibitors have been identified, and their ability to determine the activity of LMWG-regulated systems is being worked out. The discovery of multifunctional regulatory molecules has indicated that substantial "crosstalk" between various LMWG may occur. The continuing emergence of additional cellular functions that are regulated by LMWGs, and particularly the recent availability of in vitro analytical systems for studies of the mechanism (or mechanisms) of action of LMWGs, is resulting in a wealth of information about the regulation and integration of cellular signaling, form, and function.

Molecular analysis of different allelic variants of wild-type human p53

The p53 tumour suppressor gene is intensively studied because mutations in this gene are the most common genetic alteration so far identified in human cancer. Considerable emphasis has thus been placed on characterizing the biological differences between mutant and wild-type p53 protein. This has led to the realization that in cultured cells, mutant p53 behaves like an oncogene, whereas wild-type p53 is a tumor suppressor gene. The p53 protein is also a target for the tumour virus oncogene products SV40 large T, adenovirus E1B, and human papillomavirus type 16 E6, which are all capable of forming complexes to the p53 protein. Although p53 represents an extremely important cellular regulatory molecule which is well conserved, there exists two allelic variants of wild-type human p53 that differ both in primary and confirmational structure. One variant contains an arginine at amino acid 72 (p53Arg), whereas the other form contains a proline at this residue (p53Pro). The possible implications for more than one allelic variant of wild-type human p53 in the general population is unknown. The present study was undertaken to compare some of the biological features of the different wild-type p53 variants. We present data demonstrating that there was a post-transcriptional selection against accumulation of both variants of wild-type human p53 in 3T3-A31 cells, arguing that both forms are proliferation inhibitory in these cells. Both variants of human p53 were stabilized by SV40 large T, but did not displace mouse p53 from SV40 large T. Neither allelic variant of human p53 was able to reduce significantly SV40-mediated anchorage-independent growth of 3T3-A31 cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of interleukin 6 production in T helper cells

The cytokine interleukin 6 (IL-6) is a cellular regulatory molecule that is produced by both lymphoid and non-lymphoid cells in response to several stimuli. In this report we present evidence that within the murine T cell compartment T helper type 2 cells (Th2) produce this lymphokine, whereas unprimed CD4+ T cells and a T helper type 1 clone (Th1) do not. Furthermore, IL-6 is not an autocrine growth factor for in vitro cultured Th cells, in contrast to what occurs in freshly isolated CD4+ and CD8+ T cells. We have examined the signal transduction pathways that lead to IL-6 production in activated Th2 cells. We have found that protein kinase C activators, such as PMA, Con A, or IL-1, increase the IL-6 expression in these cells. On the other hand, activation of the cAMP-dependent pathway does not seem to have an effect on the IL-6 production, since forskolin, 8BrcAMP, or TNF-alpha, which in these cells increases the level of intracellular cAMP, do not lead to an accumulation of IL-6 message. These results indicate that the IL-6 gene is more tightly regulated in T cells than in other systems described previously.

Interleukin-6 is produced by bone and modulated by parathyroid hormone.

Interleukin-6 (IL-6) is a cellular regulatory molecule, the diverse functions of which relate to cells both within and outside the immune system. In this report we demonstrated that bone tissue, specifically osteoblasts, produce interleukin-6 and that this function can be modulated by the osteotrophic hormone parathyroid hormone (PTH). Given that the complex process of bone remodeling is now thought to be regulated not only by systemic hormones but also by locally produced factors, the existence of a parathyroid hormone-stimulated production of interleukin-6 by osteoblasts may have important physiological significance.

Regulation of the expression of IL-6 in human monocytes.

IL-6 is a cellular regulatory molecule with various cell-dependent functions. We have studied the control of IL-6 expression in human monocytes because they play a key role in the production of this molecule. The effects of adherence and different cytokines including CSF-1, IFN-gamma, IL-1 alpha, and granulocyte-macrophage-CSF were tested on IL-6 expression. IL-6 mRNA was usually not detected in the starting population of PBMC. Adherence induced IL-6 gene expression in monocytes in less than 2 h and subsequently IL-6 secretion. Priming of monocytes by adherence was more efficient for IL-6 overinduction by CSF-1. In contrast, high level induction of IL-6 by IFN-gamma in unfractionated PBMC did not require adherence and in situ hybridization revealed that IL-6 mRNA was present in monocytes but not in lymphocytes. A similar phenomenon was observed for IL-1 alpha and granulocyte-macrophage-CSF. Two cell lines, HL-60 and U937, in which monocytic differentiation occurs after induction by PMA, were subsequently investigated. IL-6 was not constitutively detectable in these two cell lines, whereas PMA treatment induced IL-6 expression. This effect was rapid (30 min) and transitory in HL-60, whereas IL-6 mRNA was still detected after 72 h of induction in U937. Addition of human rIL-6 on U937 and HL-60 cells inhibited their proliferation and enhanced expression of HLA class I Ag.

Isolation of genes encoding proteins of immunological importance

Publisher Summary This chapter focuses on the isolation of lymphokine genes. The chapter describes the general methodological strategies employed to isolate gene clones for important cellular regulatory molecules. The isolation of genes encoding lymphokines has been particularly challenging, requiring recently developed techniques. Lymphokines are typically low-abundance tissue-specific proteins. Therefore, the isolation of genes encoding them is hampered by a corresponding low abundance of their mRNAs: the starting material for synthesizing cDNA clones. Two important developments have increased the efficiency of identifying cDNA clones of lymphokine genes. First, biologically active lymphokines can often be synthesized by the injection of the appropriate mRNAs into Xenopus oocytes. Second, lymphokine activity can often be recovered from cells transfected with lymphokine cDNA clones placed under the regulatory control of a heterologous gene promoter. Thus, highly efficient biological methods have been developed to determine the identity of lymphokine mRNAs and cDNAs.