Table Of ContentThis work is dedicated to the generations of my family; those
that are past, those that are present, and, hopefully, those
that are future.
Glyceraldehyde-3-
Phosphate
Dehydrogenase
(GAPDH)
The Quintessential Moonlighting Protein in
Normal Cell Function and in Human Disease
Michael A. Sirover
Lewis Katz School of Medicine, Temple University
Philadelphia, PA, United States
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Biography
Michael A. Sirover, PhD, is a professor of Pharmacology, Lewis Katz School of
Medicine at Temple University, Philadelphia, PA. He received his BS in Biology
from Rensselaer Polytechnic Institute, his PhD from the State University of
New York at Stony Brook, and was a postdoctoral fellow at the Fox Chase
Cancer Center in Philadelphia. He was an associate editor of the journal Cancer
Research and, for over a decade, was the chair of a National Cancer Institute
Special Advisory Committee on Cancer Prevention.
Dr. Sirover is one of the pioneers in the identification and characterization of
multifunctional proteins. His early work on glyceraldehyde-3-phosphate dehy-
drogenase (GAPDH) helped establish it as the prime example of this new class
of cell proteins. His studies focused on its proliferative-dependent regulation,
including distinctive changes in its subcellular localization as a function of the
cell cycle, its proliferative-dependent transcriptional and translational regula-
tion, its role in DNA repair, the pathology of age-related neurodegenerative dis-
ease, and the cellular phenotype of Bloom’s syndrome, a cancer protein human
genetic disorder. He isolated and characterized anti-GAPDH monoclonal anti-
bodies and the human GAPDH gene, each of which was subsequently used by
many other researchers in their individual GAPDH studies. Lastly, he is the
author of the definitive reviews of GAPDH structure and function as well as its
role in the pathology of human disease.
Michael A. Sirover
xiii
Acknowledgments
Words cannot express my gratitude to my wife, Harlene (Lenie), without whose
support, every day and every night, I could not have written this work and to
my daughter, Jamie, for her assistance in the preparation of each chapter. Work
in the author’s laboratory was funded by grants from the National Institutes
of Health (ES-01735; CA-29414; AG14566; CA119285); the National Science
Foundation (77-20183; 8416295); and the W.W. Smith Charitable Trust.
xv
Introduction
It’s a dangerous business, Frodo, going out your door. You step onto the road, and
if you don’t keep your feet, there’s no knowing where you might be swept off to.
The Hobbit by J.R.R. Tolkien
It has been approximately three decades since I took my first step on that road
and became involved with studies on the functional diversity of glyceraldehyde-
3-phosphate dehydrogenase (GAPDH). In that time, the conception of GAPDH
has changed from a housekeeping protein of little interest to a moonlighting
protein whose structure and function are of importance not only to normal cell
function but also to the pathology of human disease. At first, studies on moon-
lighting GAPDH were met with intellectual puzzlement, curiosity, and, regret-
tably, sometimes with disdain. However, as time went on, and as study after
study demonstrated new and intriguing moonlighting GAPDH activities, the
general perception of this protein began to change. Counterintuitively, in sci-
ence, challenging conventional dogma is difficult, requiring patience, endur-
ance, and the time it takes for us to illuminate Nature’s mysteries.
This book is intended to tell the story of that decades-old journey from skep-
ticism to believability. The studies contained in it sum up our current knowledge
of the diverse roles of GAPDH, the complex protein–protein and protein–
nucleic acid interactions that underlie its moonlighting activities, as well as the
distinctive changes that occur in its subcellular localization. The book is divided
into three main sections: the first, a consideration of its role in normal cell func-
tions; the second, a discussion of its participation in the etiology of human dis-
ease; the third, a “special topics” section in which unique and novel aspects of
moonlighting GAPDH are described. In each, salient findings are included and
an overview is provided to consider how those results fit into our conception of
GAPDH as a moonlighting protein. For that purpose, in many chapters, a model
that presents a summation of the studies contained in that chapter is included.
I hope that the reader will find this endeavor to be interesting, informative, and
intriguing. Any omissions are unintentional, and the interpretation of data is
solely that of the author.
xvii
Chapter 1
The Role of Moonlighting
GAPDH in Cell Proliferation:
The Dynamic Nature of GAPDH
Expression and 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, regu-
lation, 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, cel-
lular proteins regulated actively exhibit pronounced changes in such charac-
teristics, 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 regula-
tion 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.
Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH). http://dx.doi.org/10.1016/B978-0-12-809852-3.00001-7
Copyright © 2017 Elsevier Inc. All rights reserved. 3
4 SECTION | I The Role of Moonlighting GAPDH in Normal Cell Function
1. SUBCELLULAR LOCALIZATION OF MOONLIGHTING
GAPDH DURING CELL PROLIFERATION
The active regulation of GAPDH as a function of cell proliferation was exam-
ined initially by immunocytochemical and subcellular fractionation analyses.
As illustrated in Fig. 1.1A, immunological determination of GAPDH in conflu-
ent, noncycling human fibroblasts revealed its cytosolic, nonnuclear localiza-
tion (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 prolif-
eration, 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 prolif-
erating 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 move-
ment 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
FIGURE 1.1 Subcellular GAPDH localization in noncycling cells. (A) Reprinted by permission
of Wiley and Sons. (B, C) Elsevier.
The Role of Moonlighting GAPDH in Cell Proliferation Chapter | 1 5
FIGURE 1.2 Subcellular GAPDH localization during cell proliferation. Reprinted by permission
of Wiley and Sons.
increase in GAPDH immunofluorescence in the perinuclear and nuclear regions
in proliferating cells as compared with that observed in those regions in non-
cycling cells (Fig. 1.1). Further, as cell proliferation diminished, there was a
progressive decline in immunofluorescent intensity to that observed in conflu-
ent cells (Cool and Sirover, 1989).
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 anti-
genically 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
6
S
E
C
TABLE 1.1 Immunocytochemical Localization of Moonlighting GAPDHa TI
O
N
Organism/ Experimental “Unique” I|
Cell Line Paradigm Change in GAPDH Subcellular Localization Findings References
T
h
e
Normal human Subculture of Cytoplasmic → perinuclear → nuclear → perinuclear → cytoplasmic Reversible Cool and Sirover R
fibroblasts confluent cells; subcellular (1989) and Sirover ole
asynchronous translocation of (1997) o
cell proliferation GAPDH f M
o
o
Rat liver Partial Cytoplasmic → nuclear Increase Corbin et al. n
hepatectomy in nuclear (2002) lig
h
GAPDH-no tin
change in g
G
cytoplasm A
P
D
HeLa cells Actively Nuclear Subcellular Arutyunova et al. H
proliferating localization (2003, 2013) in
of nonnative N
GAPDH orm
a
Human lung G1/S thymidine Nuclear → perinuclear → cytoplasmic S phase, G2/M Sundararaj et al. l C
adenocarcinoma block cell cycle (2004) e
specific nuclear ll F
u
localization n
c
aChronological order. tion
