Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH)

The Quintessential Moonlighting Protein in Normal Cell Function and in Human Disease

Michael A. Sirover

• Publisher: Academic Press
• Year: 2017
• Print ISBN:
  9780128098523
• eBook ISBN:
  9780128098981
• Topic: Biological Sciences
• Subtopic: Biochemistry
• Index: Biological Sciences

Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH): The Quintessential Moonlighting Protein in Normal Cell Function and in Human Disease examines the biochemical protein interactions of the multi-dimensional protein GAPDH, further considering the regulatory mechanisms through which cells control their functional diversity. This protein's diverse activities range from nuclear tRNA export and the maintenance of genomic integrity, to cytoplasmic post-transcriptional control of gene expression and receptor mediated cell signaling, to membrane facilitation of iron metabolism, trafficking and fusion. This book will be of great interest to basic scientists, clinicians and students, including molecular and cell biologists, immunologists, pathologists and clinical researchers who are interested in the biochemistry of GAPDH in health and disease. - Contextualizes how GAPDH is utilized by cells in vivo - Provides detailed insight into GAPDH post-translational modifications, including functional diversity and its subcellular localization - Includes forward-thinking exposition on tough topics, such as the exploration of how GAPDG performs functions, how it decides where it should be present and requisite structural requirements

Section I

The Role of Moonlighting GAPDH in Normal Cell Function

Outline

Chapter 1. The Role of Moonlighting GAPDH in Cell Proliferation: The Dynamic Nature of GAPDH Expression and Subcellular Localization

Chapter 2. Moonlighting GAPDH and the Transcriptional Regulation of Gene Expression: Multiprotein Complex Formation and Mechanisms of Nuclear Translocation

Chapter 3. The Diversity of Moonlighting GAPDH Function in Posttranscriptional RNA Regulation: mRNA Stability, tRNA Processing, and Viral Pathogenesis

Chapter 4. The Role of Moonlighting GAPDH in Membrane Structure and Function: Membrane Fusion and Iron Metabolism

Chapter 5. The Role of Moonlighting GAPDH in Intracellular Membrane Trafficking: Tubulin Regulation and Modulation of Cytoskeletal Structure

Chapter 6. Moonlighting GAPDH and the Maintenance of DNA Integrity: Preservation of Genetic Information and Cancer Facilitation

Chapter 7. Moonlighting GAPDH in Neuronal Structure and Function: Regulation of Ion Channels and Signal Transduction

Chapter 1

The Role of Moonlighting GAPDH in Cell Proliferation

The Dynamic Nature of GAPDH Expression and Subcellular Localization

Abstract

The distinction between housekeeping genes and those genes that are actively regulated may be defined by several criteria. Among these may be the dependence of the latter on the proliferative state of the cell, especially with respect to their subcellular localization, transcription, biosynthesis, and interactions with other cellular constituents. Recent studies suggest that moonlighting GAPDH exhibits defined changes in its expression as a function of cell proliferation. These include its cytosolic localization in noncycling cells as contrasted with its perinuclear or nuclear localization in cycling cells; its cytoplasmic relocalization as cell proliferation diminishes; enhancement of both GAPDH mRNA and protein levels in cycling cells; the physical association of GAPDH with replicating DNA; and its dissociation from the latter as cell growth diminishes. Further analysis suggests not only that GAPDH may regulate cell cycle transition but also the initiation of cell senescence. In toto, these findings suggest a complex role for moonlighting GAPDH during cell proliferation.

Keywords

Cell cycle checkpoints; Cell senescence; DNA replication; Gene expression; Glyceraldehyde-3-phosphate dehydrogenase; Subcellular localization

“If it looks like a duck, if it quacks like a duck, if it walks like a duck, it’s a duck”—a humorous term for a form of abductive reasoning

Wikipedia

A housekeeping protein may be defined as a molecule whose activity, regulation, expression, and subcellular localization remain relatively constant despite changes in cell status (growth, genome expression, environmental stress, etc.). This is reflected in their use as controls in studies quantitating cellular changes that occur in the given situation of interest. In contrast, cellular proteins regulated actively exhibit pronounced changes in such characteristics, which may be of interest in themselves and which would preclude their characterization as a housekeeping gene and protein.

For that reason, it may be suggested that the analyses of GAPDH regulation during cell proliferation may provide perhaps one of the best illustrations not only of its disqualification as a simple, classical housekeeping protein but also its designation as an active, moonlighting protein of considerable significance. In particular, these studies demonstrate pronounced, reversible changes in its intracellular localization as a function of cell growth, cell cycle changes in the transcription of GAPDH mRNA and its translation into protein, its proliferative-dependent physical association with replicating DNA, its requirement for cell cycle transition, and its role in the initiation of cell senescence. In toto, these findings, in accord with those provided in other chapters, cement GAPDH as a moonlighting protein whose function is required not only for normal cell function but also, as discussed in ensuing chapters, its role in the pathology of human disease.

1. Subcellular Localization of Moonlighting GAPDH During Cell Proliferation

The active regulation of GAPDH as a function of cell proliferation was examined initially by immunocytochemical and subcellular fractionation analyses. As illustrated in Fig. 1.1A, immunological determination of GAPDH in confluent, noncycling human fibroblasts revealed its cytosolic, nonnuclear localization (Cool and Sirover, 1989; Sirover, 1997). The recognition of the GAPDH protein was uniform in the former as was its absence in the latter. Analysis of its intracellular localization demonstrated that the overwhelming majority of immunoreactive GAPDH was present not only in the cytosol, membrane, and perinuclear regions as defined both by immunoblot analysis (Fig. 1.1B) and, as indicated in Fig. 1.1C, by comparison with the amount of immunoreactive GAPDH present in a crude cell extract (Mazzola and Sirover, 2005).

In contrast, as illustrated in Fig. 1.2 subsequent to the initiation of cell proliferation, two distinct changes were observed in human fibroblasts: the first was a change in the subcellular distribution of the GAPDH immunoreactive protein; the second was an increase in the level of GAPDH immunofluorescent staining (Cool and Sirover, 1989; Sirover, 1997). With respect to the former, in proliferating human fibroblasts, immunoreactive GAPDH exhibited a perinuclear or nuclear localization. In addition, these intracellular changes in human fibroblast immunoreactive GAPDH appeared to display a defined temporal sequence. As cell growth commenced, there was a cytoplasmic → perinuclear → nuclear movement of immunoreactive GAPDH. In contrast, as cell growth diminished and ultimately stopped, there was a nuclear → perinuclear → cytoplasmic change in immunoreactive GAPDH intracellular localization (Cool and Sirover, 1989). With respect to the latter, as indicated in Fig. 1.2, there was a considerable increase in GAPDH immunofluorescence in the perinuclear and nuclear regions in proliferating cells as compared with that observed in those regions in noncycling cells (Fig. 1.1). Further, as cell proliferation diminished, there was a progressive decline in immunofluorescent intensity to that observed in confluent cells (Cool and Sirover, 1989).

Figure 1.1 Subcellular GAPDH localization in noncycling cells. (A) Reprinted by permission of Wiley and Sons. (B, C) Elsevier.

Figure 1.2 Subcellular GAPDH localization during cell proliferation. Reprinted by permission of Wiley and Sons.

As indicated in Table 1.1, these proliferative-dependent changes in GAPDH subcellular distribution and its increased expression appeared to be a general property of growing cells.

Using partial hepatectomy as an experimental paradigm, the proliferative-dependent intracellular localization of GAPDH was determined by immunoblot analysis subsequent to subcellular fractionation (Corbin et al., 2002). In those studies, the nuclear content of GAPDH was increased 3-fold at 24 h, and the level of GAPDH mRNA was increased 1.5-fold following surgery. These two findings suggest that there was an increase in the biosynthesis of the GAPDH protein during hepatocyte cell proliferation. In contrast, the cytoplasmic level of immunoreactive GAPDH protein remained constant. This latter finding would suggest that the newly synthesized GAPDH protein could be selectively located in the nucleus. This will be considered later (Lee and Sirover, 1989).

Previous studies identified a denatured GAPDH isoform, which was antigenically distinct from the native GAPDH isoform (Grigorieva et al., 1999). Recently, using that antibody, which specifically recognized the denatured GAPDH species, its intracellular localization was probed in HeLa cells (Arutyunova et al., 2003, 2013). These studies demonstrated that denatured GAPDH exhibited a nuclear localization during cell proliferation. However, in contrast to native GAPDH, its nuclear localization did not appear to be evenly distributed. This suggested that the denatured form may provide moonlighting functions distinct from those observed with native GAPDH. Evidence was also presented indicating a cytoplasmic colocalization with ac...

Table of contents

• Cover image
• Title page
• Table of Contents
• Dedication
• Copyright
• Biography
• Acknowledgments
• Introduction
• Section I. The Role of Moonlighting GAPDH in Normal Cell Function
• Section II. Physiological Stress and GAPDH Functional Diversity
• Section III. The Pathology of GAPDH Functional Diversity
• Section IV. The Pharmacology of Moonlighting GAPDH
• Section V. The Unique Role of Sperm-Specific GAPDH
• Section VI. Discussion
• Index