A Perspective on Personalized Medicine
Curator: Larry H. Bernstein, MD, FCAP
A book has recently been reviewed by Laura Fisher (Feb 19 2016) titled “Junk DNA: a journey through the dark matter of the genome” (Nessa Carey Icon Books 2015 | 352pp ISBN 9781848319158). http://www.rsc.org/chemistryworld/2016/02/junk-dna-genome-nessa-carey-book-review It is important in its focus on, ‘junk DNA’, a term coined in the 1960s that refers to regions of our DNA that don’t code for proteins. It is now known that a large portion of the genome is noncoding. These non-coding areas of our DNA are far from being without function. Whether regulating gene expression and transcription, or providing protein attachment sites, this once-dismissed part of the genome is vital for all life, and this is the focus of Junk DNA. However, in 1869 Friedrich Miescher discovered a new substance (Dahm, 2008) from the cell nuclei that had chemical properties unlike any protein, including a much higher phosphorous content and resistance to proteolysis (protein digestion). He wrote, “It seems probable to me that a whole family of such slightly varying phosphorous-containing substances will appear, as a group of nucleins, equivalent to proteins” (Wolf, 2003). In 1971, Chargaff noted that Miescher’s discovery of nucleic acids was unique among the discoveries of the four major cellular components (i.e., proteins, lipids, polysaccharides, and nucleic acids) in that it could be “dated precisely… [to] one man one place, one date.” We now know that there are two basic categories of nitrogenous bases: the purines (adenine [A] and guanine [G]), each with two fused rings, and the pyrimidines (cytosine [C], thymine [T], and uracil [U]), each with a single ring. Furthermore, it is now widely accepted that RNA contains only A, G, C, and U (no T), whereas DNA contains only A, G, C, and T (no U). Keeping this in mind, the Watson-Crick proposal, as important as it was, was a discovery out of historical proportion, and it set the path of molecular biology for the remainder of the 20th century. A consequence of this seminal event was that the direction of biochemistry and molecular biology became set for several generations into the 21st century, culminating in the Human Genome Project.
As important as this discovery and others related that followed, there were a number of unrelated discoveries that took on huge importance, immediately recognized, but not so soon integrated with the evolving body of knowledge. For example, since the 1920s, the work of Warburg and Meyerhoff, followed by that of Krebs, Kaplan, Chance, and others built a solid foundation in the knowledge of enzymes, coenzymes, adenine and pyridine nucleotides, and metabolic pathways, not to mention the importance of Fe3+, Cu2+, Zn2+, and other metal cofactors. There was also a relevance of the work of Jacob, Monod and Changeux, and the effects of cooperativity in allosteric systems and of repulsion in tertiary structure of proteins related to hydrophobic and hydrophilic interactions. This involves the effect of one ligand on the binding or catalysis of another with no direct interaction between the two ligands. This was demonstrated by the end-product inhibition of the enzyme, L-threonine deaminase (Changeux 1961), L-isoleucine, which differs sterically from the reactant, L-threonine whereby binding at a different, nonoverlapping (regulatory) site, the former could inhibit the enzyme without competing with the latter. Pauling (Pauling 1935) had earlier proposed a model for intramolecular control in hemoglobin to explain the positive cooperativity observed in the binding of oxygen molecules. But Monod, Wyman, and Changeux substantially updated the view of allostery in 1965 with their landmark paper. Present day applications of computational methods to biomolecular systems, combined with structural, thermodynamic, and kinetic studies, make possible an approach to that question, so as to provide a deeper understanding of the requirements for allostery. The current view is that a variety of measurements (e.g., NMR, FRET, and single molecule studies) are providing additional data beyond that available previously from structural, thermodynamic, and kinetic results. These should serve to continue to improve our understanding of the molecular mechanism of allostery, particularly when supplemented by simulations and theoretical analyses. A ‘‘dynamic’’ proposal by Cooper and Dryden (1984) is that the distribution around the average structure changes in allostery; which in turn, affects the subsequent (binding) affinity at a distant site. Such a model focuses on the vibrational contribution to the entropy as the origin of cooperativity, as discussed for the CAPN dimer. Why is this important? It is because it brings a different focus into the conception of how living cells engage with their neighbors and external environment. Moreover, this is not all that has to be considered.
What else do we have to consider? Oxidative stress is essentially an imbalance between the production of free radicals and the ability of the body to counteract or detoxify their harmful effects through neutralization by antioxidants. The measurement of free radicals has increased awareness of radical-induced impairment of the oxidative/antioxidative balance, essential for an understanding of disease progression. Metal-mediated formation of free radicals causes various modifications to DNA bases, enhanced lipid peroxidation, and altered calcium and sulfhydryl homeostasis. Lipid peroxides, formed by the attack of radicals on polyunsaturated fatty acid residues of phospholipids, can further react with redox metals finally producing mutagenic and carcinogenic malondialdehyde, 4-hydroxynonenal and other exocyclic DNA adducts (etheno and/or propano adducts). The unifying factor in determining toxicity and carcinogenicity for all these metals is the generation of reactive oxygen and nitrogen species. Common mechanisms involving the Fenton reaction, generation of the superoxide radical and the hydroxyl radical appear to be involved for iron, copper, chromium, vanadium and cobalt primarily associated with mitochondria, microsomes and peroxisomes. Various studies have confirmed that metals activate signaling pathways and the carcinogenic effect of metals has been related to activation of mainly redox sensitive transcription factors, involving NF-kappaB, AP-1 and p53.
In addition to what I have identified, there is substantial work in the last decade to indicate a more complex model of cellular regulatory processes. On the one hand, there is no uncertainty about the importance of “Junk DNA”. Indeed, not only is “Junk DNA” not junk, but it has either a presence that is an evolutionary remnant, or it has a role in cell regulation, much of which has yet to be understood. Moreover, the relationship between the oligonucleotide sequences to their histones are largely unknown. Beyond the DNA sequences, the language of the gene, we now have a large output of research on noncoding RNA. We now have siRNA, miRNA, and others with roles other than transcription. This is a very active field of investigation that requires major revision of our model of cell regulatory processes. The classic model is solely transcriptional. DNA-> RNA-> Amino Acid in a protein. This would now have to be redrawn because DNA-> RNA-> DNA and DNA->RNA-> protein-> DNA.
I have provided a series of four mechanisms explanatory for transcription and for regulation of the cell. This is not adequate for a more full comprehension because there is a layer beyond the classic model of metabolic pathways associated with the cytoplasm, mitochondria, endoplasmic reticulum, and lysosome, there are critical paths beyond oxidative phosphorylation and glycolysis, such as the cell death pathways, expressed in a homeostasis between apoptosis and repair. Nevertheless, there is still a missing part of this discussion. The missing piece gets at the time and space interactions of the cell, cellular cytoskeleton and extracellular and intracellular substrate interactions in the immediate environment. This can’t be simply accounted for by genetics or epigenetics. There have been papers that call attention to heterogeneity among cancer cells of expected identical type, which would be consistent with differences in phenotypic expression, aligned with epigenetics. There is now the recent publication of the finding that there is heterogeneity in the immediate interstices between cancer cells, which may seem surprising, but it should not be. This refers to the complexity of the cells arranged as tissues and to their immediate environment, which I shall elaborate on. Integration with genome-wide profiling data identified losses of specific genes on 4p14 and 5q13 that were enriched in grade 3 tumors with high microenvironmental diversity that also substratified patients into poor prognostic groups. I did introduce the word gene into this reference, and we are well aware of mutations that occur in cancer progression. In the case of breast cancer, mention is not made of interaction with a hormone, as we refer to in androgen-unresponsive prostate cancer. This is particularly relevant, but incomplete.
The fifth item for discussion is the interaction between enzyme and substrates that may be conditionally unidirectional in defining the activity within the cell. When we speak of the genome, we are dealing with a code defined by an oligonucleotide sequence that has an element of stability, but that can conditionally be altered by a process termed mutagenesis. The altered code can be expected to have a negative, positive, or no effect, depending. In any case, there is a substantial stability inherent in the code that is essential to all living creatures. The activity of the cell is dynamically interacting and at high rates of activity. There are many examples of this. The first example is in a study of energy for reverse pyruvate kinase (PK) reaction. This catalytic activity of the PK reaction was reversed to the thermodynamically unfavorable direction in a muscle preparation by a specific inhibitor. Using the same crude supernatant for the two opposite activities of this enzyme some of the results found in the regulatory assays indicated differences in the active form of pyruvate kinase that were clearly related to the environmental condition – glycolitic or glyconeogenetic – of the assay. The conformational changes indicated by differential regulatory response found in the conditions studied, together with the role of similar factors, for instance, substrates and pH, in the structural states proposed by others, were used together to present a dynamic conformational model functioning at the active site of the enzyme. In the model, the interaction of the enzyme active site with its substrates is described according to its vibrational, translational and rotational components and the activating ions – induced increase in the vibrational energy levels of the active site decreases the energetic barrier for substrate induced changes at the site.
Another example is the pyridine nucleotide-linked dehydrogenases. The lactate dehydrogenase (LD) reaction is ordered so that NADH binds to the enzyme before pyruvate can bind. The H-type isoenzyme, but not the M-type, is characterized by substrate inhibition at high pyruvate concentrations. The inhibition of the H4 lactate dehydrogenase, but not the M4, by high concentrations of pyruvate is caused by the formation of an abortive complex consisting of the enzyme, pyruvate, and NADH. An investigation of the structural properties of the ternary complex revealed that the complex possesses an absorption maximum at 335 nm and that a covalent bond was formed between the nicotinamide ring of the NAD+ and the pyruvate moiety. The same study demonstrated that the enol form of pyruvate is responsible for the complex formation. It was suggested that abortive complex formation is a significant metabolic control mechanism, and the different behavior of the H and M forms has been rationalized in terms of different functional roles for the two isoenzymes. However, similar experiments carried out with the mitochondrial malate dehydrogenase suggested a similar inhibition, but in this case only the mitochondrial malate dehydrogenase is sensitive to inhibition by high concentrations of oxaloacetate. Further studies showed the inhibition is promoted by an abortive binary complex formed by the enzymes and the enol form of oxalacetate. Neither the oxidized coenzyme nor the reduced coenzyme appears to be involved in the formation of this complex. These results suggest that the mechanism of substrate inhibition that occurs with the pig heart malate dehydrogenases is different from that observed with the lactate dehydrogenases.
It was established years later that there is an isoenzyme of isocitrate dehydrogenase that is characteristic for cancer cells. IDH1 and IDH2 mutations occur frequently in some types of World Health Organization grades 2–4 gliomas and in acute myeloid leukemias with normal karyotype. IDH1 and IDH2 mutations are remarkably specific to codons that encode conserved functionally important arginines in the active site of each enzyme. To date, all IDH1 mutations have been identified at the Arg132 codon. Mutations in IDH2 have been identified at the Arg140 codon, as well as at Arg172, which is aligned with IDH1 Arg132. IDH1 and IDH2 mutations are heterozygous in cancer, and they catalyze the production of α-2-hydroxyglutarate. The study found human IDH1 transitions between an inactive open, an inactive semi-open, and a catalytically active closed conformation. In the inactive open conformation, Asp279 occupies the position where the isocitrate substrate normally forms hydrogen bonds with Ser94. This steric hindrance by Asp279 to isocitrate binding is relieved in the active closed conformation.
Finally, what does this have to do with personalized medicine? Personalized medicine has been largely view from a lens of genomics. But genomics is only the reading frame, even taking into consideration the mutations that are found in transition. The living activities of cell processes are dynamic and occur at rapid rates. When we refer to homeostasis and to neoplasia, we have to keep in mind that personalized in reference to genotype is not complete without reconciliation of phenotype, which is the reference to expressed differences in outcomes.
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