Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology
ReviewNew insights into an old protein: the functional diversity of mammalian glyceraldehyde-3-phosphate dehydrogenase
Introduction
“Nature is nowhere accustomed more openly to display her secret mysteries than in the cases where she shows traces of her work apart from the beaten path.” William Harvey
The purpose of this review is to consider new, recent studies which suggest the need to reinterpret both the structure and the function of the protein glyceraldehyde-3-phosphate dehydrogenase (GAPDH, EC 1.2.1.12) in mammalian cells. For over three decades, GAPDH was studied for its pivotal role in glycolysis. As an abundant cell protein, it proved useful as a model for investigations examining basic mechanisms of enzyme action as well as the relationship between amino acid sequence and protein structure. Further, with the advent of molecular technology, GAPDH, as a putative ‘house-keeping’ gene, provided a model with which to use new methods for gene analysis to advance our understanding of the mechanisms through which cells organize and express their genetic information.
As with many things in life, what is thought to be simple and relatively straight-forward turns out to be quite complex and elaborate. In this regard, a number of studies, accelerating in the last decade [1], have indicated that GAPDH is not an uncomplicated, simple glycolytic protein. Instead, as illustrated in Fig. 1, independent laboratories identified diverse biological properties of the mammalian GAPDH protein. These included roles for GAPDH in membrane transport and in membrane fusion, microtubule assembly, nuclear RNA export, protein phosphotransferase/kinase reactions, the translational control of gene expression, DNA replication and DNA repair. Each activity appears to be distinct from its glycolytic function.
Other independent studies implicate GAPDH as an essential part of the program of gene expression observed in apoptosis and as part of the cellular phenotype of age-related neuronal disorders. In the former, this may include nuclear translocation of GAPDH into the nucleus without the induction of nuclear GAPDH glycolytic activity. In the latter, this may include the physical association of GAPDH with the β-amyloid precursor protein in Alzheimer’s disease and with proteins involved in ‘gain of function’ CAG triplet repeat disorders. Drugs which inhibit apoptosis and are currently in use to treat Parkinson’s disease specifically bind to GAPDH. Recent evidence also suggests a role for GAPDH in the etiology of prostate cancer. This may involve the dysregulation of GAPDH gene expression and the appearance of new GAPDH isoforms. Lastly, other laboratories have indicated a relationship between GAPDH and the pharmacology and toxicology of nitric oxide. NO induces oxidatively the covalent binding of NAD+ to GAPDH and, by S-nitrosylation, inhibits its dehydrogenase activity. At present, it does not appear that any other mammalian protein is so modified by nitric oxide.
Accordingly, the focus of this review is to consider the structure of the GAPDH protein as it relates to the new and novel functions identified for it in multiple reports from independent investigators. The basic rationale which will be followed is to discuss the functional diversity of GAPDH. Subsequently, reinterpretation of GAPDH protein structure will be addressed to postulate mechanisms through which classically defined GAPDH amino acid sequences may now be used in mammalian cells for such non-glycolytic activities. GAPDH genetic organization and protein structure will be discussed in relation to its role in fundamental biological processes (i.e., apoptosis) or pathologies (i.e., neuronal disorders). In addition, agents (i.e., nitric oxide, glutathione, the β-amyloid protein, the Huntington protein) which might affect GAPDH structure could dramatically alter its function. Considering its multidimensional nature, such alterations in GAPDH structure and function would induce pleiotropic changes in mammalian cells.
Section snippets
Functional diversity of mammalian GAPDH
The multidimensional nature of GAPDH is based on a series of new studies by independent laboratories. For the purposes of this section, these recent investigations will be grouped into two categories. The first group includes studies which identify new activities of GAPDH. As such, they provide documentation of specific functions apart from its conventional dehydrogenase activity. The second group includes new studies which identify the specific binding between GAPDH and cellular
Structure–function analysis of GAPDH: non-glycolytic activities
As described in the previous section, mammalian GAPDH exhibits a variety of functions distinct from its classical glycolytic dehydrogenase activity. In particular, the emerging understanding of its multifunctional activities, subcellular localization and potential role in apoptosis, neuronal disorders and prostate cancer requires new molecular studies evaluating its nucleotide and amino acid sequence as well as its protein–protein interactions in relation to its non-glycolytic functions.
Organization and expression of the mammalian GAPDH gene
In previous sections we discussed the multiple activities of mammalian GAPDH. We considered as well mechanisms through which its conventional glycolytic structure may have been adapted to these new functions. In this section, we intend to first review the genetic organization and expression of mammalian GAPDH. Second, we will then introduce studies which present an apparent paradox with respect to the interrelationship between GAPDH gene structure and its functional diversity.
Nitric oxide/GAPDH interactions
Nitric oxide exhibits a duality of function in mammalian cells. First, it is a critical intracellular compound which functions as an in vivo messenger within a number of cell processes including inflammation, the immune response and acts as a neurotransmitter (reviewed in [122], [123]). Initial studies identified cGMP-dependent pathways mediated by guanylate cyclase as basic molecular mechanisms for NO function. Other studies indicated a role for cGMP independent pathways in the mechanisms of
Role of GAPDH in apoptosis
Initial studies in cerebral granular cells (CGCs) first demonstrated the role of GAPDH in apoptosis [139], [140], [141], [142], [143]. CGCs in culture underwent apoptosis at approximately 17 days in vitro (DIV). SDS–PAGE analysis revealed the appearance of a 38 kDa protein coordinate with the induction of apoptosis. Actinomycin D and cycloheximide reduced expression of the unknown protein. N-Terminal sequence analysis identified the protein as GAPDH. The physiological relevance of GAPDH
GAPDH and neurodegenerative disease: protein–protein interactions
As indicated above, recent studies provide significant evidence for the role of GAPDH in apoptosis. GAPDH participation in this basic cellular process may be interrelated with its role in human pathology. This is suggested by several independent studies which indicate a specific relationship in vivo between GAPDH and a number of proteins directly related to the pathology of age-related human neuronal disorders. These include specific protein–protein interactions between GAPDH and the β-amyloid
GAPDH and prostate cancer
Investigations on the role of GAPDH in the cellular phenotype of prostate cancer focused on alterations of GAPDH gene and protein expression. In these studies prostate cancer cell lines as well as prostate tissue were examined. The rationale was that, were GAPDH to play a role in prostate cancer, it would be reasonable to detect transcriptional or translational aberrations as compared to normal cells.
Initial studies quantitated GAPDH mRNA levels in rat adenocarcinoma cell lines in comparison
Conclusions
The state of our knowledge with respect to the functional diversity of mammalian GAPDH and the underlying basic mechanisms which provide for its multidimensional nature is still in its infancy. It may be accurate to say that there are many questions which may be posed with respect to GAPDH structure and function. It is also clear that few answers are currently available to respond to those queries. Accordingly, fertile ground exists for future experimentation. Several areas appear promising.
Acknowledgements
The generous assistance of Dr. Jerome L. Gabriel in molecular modeling analysis is gratefully acknowledged. Work in the author’s laboratory was funded by a grant from the National Institute on Aging (AG14566).
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