Posts Tagged ‘LDH isoenzymes’

A Reconstructed View of Personalized Medicine

Author: Larry H. Bernstein, MD, FCAP


There has always been Personalized Medicine if you consider the time a physician spends with a patient, which has dwindled. But the current recognition of personalized medicine refers to breakthrough advances in technological innovation in diagnostics and treatment that differentiates subclasses within diagnoses that are amenable to relapse eluding therapies.  There are just a few highlights to consider:

  1. We live in a world with other living beings that are adapting to a changing environmental stresses.
  2. Nutritional resources that have been available and made plentiful over generations are not abundant in some climates.
  3. Despite the huge impact that genomics has had on biological progress over the last century, there is a huge contribution not to be overlooked in epigenetics, metabolomics, and pathways analysis.

A Reconstructed View of Personalized Medicine

There has been much interest in ‘junk DNA’, non-coding areas of our DNA are far from being without function. DNA has two basic categories of nitrogenous bases: the purines (adenine [A] and guanine [G]), and the pyrimidines (cytosine [C], thymine [T], and  no uracil [U]),  while RNA contains only A, G, C, and U (no T).  The Watson-Crick proposal set the path of molecular biology for decades into the 21st century, culminating in the Human Genome Project.

There is no uncertainty about the importance of “Junk DNA”.  It is both an evolutionary remnant, and it has a role in cell regulation.  Further, the role of histones in their relationship the oligonucleotide sequences is not understood.  We now have a large output of research on noncoding RNA, including siRNA, miRNA, and others with roles other than transcription. This requires major revision of our model of cell regulatory processes.  The classic model is solely transcriptional.

  • DNA-> RNA-> Amino Acid in a protein.

Redrawn we have

  • DNA-> RNA-> DNA and
  • DNA->RNA-> protein-> DNA.

Neverthess, there were unrelated discoveries that took on huge importance.  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.  Of huge importance was 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, which involves the effect of one ligand on the binding or catalysis of another,  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 the former could inhibit the enzyme without competing with the latter. The current view based on a variety of measurements (e.g., NMR, FRET, and single molecule studies) is a ‘‘dynamic’’ proposal by Cooper and Dryden (1984) that the distribution around the average structure changes in allostery affects the subsequent (binding) affinity at a distant site.

What else do we have to consider?  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. 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.

I have provided mechanisms explanatory for regulation of the cell that go beyond the classic model of metabolic pathways associated with the cytoplasm, mitochondria, endoplasmic reticulum, and lysosome, such as, the cell death pathways, expressed in apoptosis and repair.  Nevertheless, there is still a missing part of this discussion that considers the time and space interactions of the cell, cellular cytoskeleton and extracellular and intracellular substrate interactions in the immediate environment.

There is heterogeneity among cancer cells of expected identical type, which would be consistent with differences in phenotypic expression, aligned with epigenetics.  There is also heterogeneity in the immediate interstices between cancer cells.  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. In the case of breast cancer, there is interaction with estrogen , and we refer to an androgen-unresponsive prostate cancer.

Finally,  the interaction between enzyme and substrates may be conditionally unidirectional in defining the activity within the cell.  The activity of the cell is dynamically interacting and at high rates of activity.  In a study of the pyruvate kinase (PK) reaction the catalytic activity of the PK reaction was reversed to the thermodynamically unfavorable direction in a muscle preparation by a specific inhibitor. Experiments found that in there were differences in the active form of pyruvate kinase that were clearly related to the environmental condition of the assay – glycolitic or glyconeogenic. The conformational changes indicated by differential regulatory response were used 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 concluding that 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 inhibition of H4 lactate dehydrogenase, but not the M4, by high concentrations of pyruvate. An investigation of the inhibition revealed that a covalent bond was formed between the nicotinamide ring of the NAD+ and the enol form of pyruvate.  The isoenzymes of isocitrate dehydrogenase, IDH1 and IDH2 mutations occur in 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. In this case, there is steric hindrance by Asp279 where the isocitrate substrate normally forms hydrogen bonds with Ser94.

Personalized medicine has been largely viewed from a lens of genomics.  But genomics is only the reading frame.  The living activities of cell processes are dynamic and occur at rapid rates.  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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More Complexity in Protein Evolution

Author and Curator: Larry H. Bernstein, MD, FCAP 

Lactate dehydrogenase like crystallin: a potentially protective shield for Indian spiny-tailed lizard (Uromastix ltardwickit) lens against environmental stress?
A Atta, A Ilyas, Z Hashim, A Ahmed and S Zarina
The Protein Journal 2014; 33(2), p. 128-34.

Taxon specific lens crystallins in ve1iebrates are either similar or identical with various metabolic enzymes. These bifunctional crystallins serve as structural protein in lens along with their catalytic role. In the present study, we have partially purified and characterized lens crystallin from Indian spiny-tailed lizard (Uroma stix hardwickii). We have found lactate dehydrogenase (LDH) activity in lens indicating presence of an enzyme crystallin with dual functions. Taxon specific lens crystallins are product of gene sharing or gene duplication phenomenon where a pre-existing enzyme is recruited as lens crystallin in addition to structural role. In lens, same gene adopts refractive role in lens without modification or loss of pre-existing function during gene sharing phenomenon. Apart from conventional role of structural protein, LDH activity containing crystallin in Uromastix hardwickii lens is likely to have adaptive characteristics to offer protection against toxic effects of oxidative stress and ultraviolet light, hence justifying its recruitment. Taxon specific crystallins may serve as good models to understand structure-function relationship of these proteins.

αB-Crystallin and 27-kd Heat Shock Protein Are Regulated by Stress Conditions in the Central Nervous System and Accumulate in Rosenthal Fibers
T Iwaki, A Iwaki, J Tateishi, Y Sakaki, and JE Goldmant
Ameri J Pathol  1993; 143(2):487-495.

To understand the significance of the accumulation of αB-crystallin in Rosenthal fibers within astrocytes, the expression and metabolism of αB-crystallin in glioma cell lines were examined under the conditions of heat and oxidative stress. αB-crystallin mRNA was increased after both stresses, and αB-crystallin protein moved from a detergent-soluble to a detergent-insoluble form. In addition, Western blotting of Alexander’s  disease brain homogenates revealed that the 27-kd heat shock protein (HSP27), which is related to αB-crystallin, accumulates along with αB-crystallin. The presence of HSP27 in Rosenthal fibers was directly demonstrated by immunohistochemistry. Our results suggest that astrocytes in Alexander’s disease may be involved in an as yet unknown kind of stress reaction that causes the accumulation of αB-ccystallin and HSP27 and results in Rosenthal fiber formation.

α-Crystallin can function as a molecular chaperone
Joseph Horwitz
Proc. Nadl. Acad. Sci. USA Nov 1992; 89: 10449-10453. Biochemistry

The α-crystallins (αA and αB) are major lens structural proteins of the vertebrate eye that are related to the small heat shock protein family. In addition, crystallins (especially αB) are found in many cells organs outside the lens, and aα is overexpressed in several neurological disorders and in cell lines under stress conditions. Here I show that α-crystallin can function as a molecular chaperone. Stoichiometric amounts of αA and αB suppress thermally induced aggregation of various enzymes. In particular, α-crystalln is very efficient in suppressing the thermally induced aggregation of β- and y-crystallins, the two other major mammalian stuctural lens proteins. α-Crystallin was also effective in preventing aggregation and in refolding guanidine hydrochloride-denatured y-crystallin, as judged by circular dichroism spectroscopy. My results thus indicate that α-crystallin refracts light and protects proteins from aggregation in the transparent eye lens and that in nonlens cells α-crystallin may have other functions in addition to its capacity to suppress aggregation of proteins.

Gene sharing by δ-crystallin and argininosuccinate Iyase
J Piatigorsky, WE O’Brient, BL Norman, K Kalumuckt, GJ Wistow, T Borras, et al.
Proc. Natl. Acad. Sci. USA  May 1988; 85: 3479-3483. Evolution.

The lens structural protein δ-crystallin and the metabolic enzyme argininosuccinate lyase (ASL; Largininosuccinate argine-lyase, EC have striking sequence similarity. We have demonstrated that duck δ-crystallin has enormously high ASL activity, while chicken δ-crystallin has lower but significant activity. The lenses of these birds had much greater ASL activity than other tissues, suggesting that ASL is being expressed at unusually high levels as a structural component. In Southern blots of human genomic DNA, chicken δ1-crystallin cDNA hybridized only to the human ASL gene; moreover, the two chicken δ-crystallin genes accounted for all the sequences in the chicken genome able to cross-hybridize with a human ASL cDNA, with preferential hybridization to the δ2 gene. Correlations of enzymatic activity and recent data on mRNA levels in the chicken lens suggest that ASL activity depends on expression of the δ2-crystallin gene. The data indicate that the same gene, at least in ducks, encodes two different functions, an enzyme (ASL) and a structural protein (δ-crystallin), although in chickens specialization and separation of functions may have occurred.

Gecko i-crystallin: How cellular retinol-binding protein became an eye lens ultraviolet filter
PJ L Werten, Beate Roll, DMF van Aalten, and WW de Jong
PNAS Mar 2000; 97(7): 3282–3287

Eye lenses of various diurnal geckos contain up to 12% i-crystallin. This protein is related to cellular retinol-binding protein type I (CRBP I) but has 3,4-didehydroretinol, rather than retinol, as a ligand. The 3,4-didehydroretinol gives the lens a yellow color, thus protecting the retina by absorbing short-wave radiation. i-Crystallin could be either the gecko’s housekeeping CRBP I, recruited for an additional function in the lens, or the specialized product of a duplicated CRBP I gene. The finding of the same CRBP I-like sequence in lens and liver cDNA of the gecko Lygodactylus picturatus now supports the former option. Comparison with i-crystallin of a distantly related gecko, Gonatodes vittatus, and with mammalian CRBP I, suggests that acquiring the additional lens function is associated with increased amino acid changes. Compared with the rat CRBP I structure, the i-crystallin model shows reduced negative surface charge, which might facilitate the required tight protein packing in the lens. Other changes may provide increased stability, advantageous for a long-living lens protein, without frustrating its role as retinol transporter outside the lens. Despite a number of replacements in the ligand pocket, recombinant i-crystallin binds 3,4-didehydroretinol and retinol with similar and high affinity (1.6 nM). Availability of ligand thus determines whether it binds 3,4-didehydroretinol, as in the lens, or retinol, in other tissues. i-Crystallin presents a striking example of exploiting the potential of an existing gene without prior duplication.

Expression of βA3/A1-crystallin in the developing and adult rat eye
G Parthasarathy, Bo Ma, C Zhang, C Gongora, JS Zigler, MK Duncan, D Sinha
J Molec Histol 2011; 42(1): 59-69.

Crystallins are very abundant structural proteins of the lens and are also expressed in other tissues. We have previously reported a spontaneous mutation in the rat βA3/A1-crystallin gene, termed Nuc1, which has a novel, complex, ocular phenotype. The current study was undertaken to compare the expression pattern of this gene during eye development in wild type and Nuc1 rats by in situ hybridization (ISH) and immunohistochemistry (IHC).
βA3/A1-crystallin expression was first detected in the eyes of both wild type and Nuc1 rats at embryonic (E) day 12.5 in the posterior portion of the lens vesicle, and remained limited to the lens fibers throughout fetal life.
After birth, βA3/A1-crystallin expression was also detected in the neural retina (specifically in the astrocytes and ganglion cells) and in the retinal pigmented epithelium (RPE).
This suggested that βA3/A1-crystallin is not only a structural protein of the lens, but has cellular function(s) in other ocular tissues.
In summary, expression of βA3/A1-crystallin is controlled differentially in various eye tissues with lens being the site of greatest expression.
Similar staining patterns, detected by ISH and IHC, in wild type and Nuc1 animals suggest that functional differences in the protein, rather than changes in mRNA/protein level of expression likely account for developmental abnormalities in Nuc1.

βA3/A1Crystallin controls anoikis-mediated cell death in astrocytes by modulating PI3K/AKT/mTOR and ERK survival pathways through the PKD/Bit1-signaling axis
B Ma, T Sen, L Asnaghi, M Valapala, F Yang, S Hose, D S McLeod, Y Lu, et la.
Cell Death and Disease 2011; 2(10).

During eye development, apoptosis is vital to the maturation of highly specialized structures such as the lens and retina. Several forms of apoptosis have been described, including anoikis, a form of apoptosis triggered by inadequate or inappropriate cell–matrix contacts. The anoikis regulators, Bit1 (Bcl-2 inhibitor of transcription-1) and protein kinase-D (PKD), are expressed in developing lens when the organelles are present in lens fibers, but are downregulated as active denucleation is initiated.
We have previously shown that in rats with a spontaneous mutation in the Cryba1 gene, coding for βA3/A1-crystallin, normal denucleation of lens fibers is inhibited. In rats with this mutation (Nuc1), both Bit1 and PKD remain abnormally high in lens fiber cells. To determine whether βA3/A1-crystallin has a role in anoikis, we induced anoikis in vitro and conducted mechanistic studies on astrocytes, cells known to express βA3/A1-crystallin.
The expression pattern of Bit1 in retina correlates temporally with the development of astrocytes. Our data also indicate that loss of βA3/A1-crystallin in astrocytes results in a failure of Bit1 to be trafficked to the Golgi, thereby suppressing anoikis. This loss of βA3/A1-crystallin also induces insulin-like growth factor-II, which increases cell survival and growth by modulating the phosphatidylinositol-3-kinase (PI3K)/AKT/mTOR and extracellular signal-regulated kinase pathways. We propose that βA3/A1-crystallin is a novel regulator of both life and death decisions in ocular astrocytes.

βA3/A1-crystallin in astroglial cells regulates retinal vascular remodeling during development
D Sinha, A Klise, Y Sergeev, S Hose, IA Bhutto, L Hackler Jr., T Malpic-llanos, et al.
Molec Cell Neurosci 2008; 37(1): 85-95.

Vascular remodeling is a complex process critical to development of the mature vascular system. Astrocytes are known to be indispensable for initial formation of the retinal vasculature; our studies with the Nuc1 rat provide novel evidence that these cells are also essential in the retinal vascular remodeling process.
Nuc1 is a spontaneous mutation in the Sprague–Dawley rat originally characterized by nuclear cataracts in the heterozygote and microphthalmia in the homozygote. We report here that the Nuc1 allele results from mutation of the βA3/A1-crystallin gene, which in the neural retina is expressed only in astrocytes. We demonstrate striking structural abnormalities in Nuc1 astrocytes with profound effects on the organization of intermediate filaments. While vessels form in the Nuc1 retina, the subsequent remodeling process required to provide a mature vascular network is deficient. Our data implicate βA3/A1-crystallin as an important regulatory factor mediating vascular patterning and remodeling in the retina.

A developmental defect in astrocytes inhibits programmed regression of the hyaloid vasculature in the mammalian eye
C Zhang, L Asnaghi, C Gongora, B Patek, S Hose, Bo Ma, MA Fard, L Brako, et al.
Eur J Cell Biol 2011; 90(5): 440-448.

Previously we reported the novel observation that astrocytes ensheath the persistent hyaloid artery, both in the Nuc1 spontaneous mutant rat, and in human PFV (persistent fetal vasculature) disease (Developmental Dynamics 234:36–47, 2005). We now show that astrocytes isolated from both the optic nerve and retina of Nuc1 rats migrate faster than wild type astrocytes. Aquaporin 4 (AQP4), the major water channel in astrocytes, has been shown to be important in astrocyte migration. We demonstrate that AQP4 expression is elevated in the astrocytes in PFV conditions, and we hypothesize that this causes the cells to migrate abnormally into the vitreous where they ensheath the hyaloid artery. This abnormal association of astrocytes with the hyaloid artery may impede the normal macrophage-mediated remodeling and regression of the hyaloid system.

βA3/A1-crystallin is required for proper astrocyte template formation and vascular remodeling in the retina.
D Sinha; WJ Stark; M Valapala; IA Bhutto; M Cano; S Hose; GA Lutty; et al.  Transgenic research 2012; 21(5):1033-42.

Nuc1 is a spontaneous rat mutant resulting from a mutation in the Cryba1 gene, coding for βA3/A1-crystallin. Our earlier studies with Nuc1 provided novel evidence that astrocytes, which express βA3/A1-crystallin, have a pivotal role in retinal remodeling. The role of astrocytes in the retina is only beginning to be explored. One of the limitations in the field is the lack of appropriate animal models to better investigate the function of astrocytes in retinal health and disease. We have now established transgenic mice that overexpress the Nuc1 mutant form of Cryba1, specifically in astrocytes. Astrocytes in wild type mice show normal compact stellate structure, producing a honeycomb-like network. In contrast, in transgenics over-expressing the mutant (Nuc1) Cryba1 in astrocytes, bundle-like structures with abnormal patterns and morphology were observed. In the nerve fiber layer of the transgenic mice, an additional layer of astrocytes adjacent to the vitreous is evident. This abnormal organization of astrocytes affects both the superficial and deep retinal vascular density and remodeling. Fluorescein angiography showed increased venous dilation and tortuosity of branches in the transgenic retina, as compared to wild type. Moreover, there appear to be fewer interactions between astrocytes and endothelial cells in the transgenic retina than in normal mouse retina. Further, astrocytes overexpressing the mutant βA3/A1-crystallin migrate into the vitreous, and ensheath the hyaloid artery, in a manner similar to that seen in the Nuc1 rat. Together, these data demonstrate that developmental abnormalities of astrocytes can affect the normal remodeling process of both fetal and retinal vessels of the eye and that βA3/A1-crystallin is essential for normal astrocyte function in the retina.

Ontogeny of oxytocin and vasopressin receptor binding in the lateral septum in prairie and montane voles
Z. Wang, L.J. Young
Developmental Brain Research 1997; 104:191–195.

Adult prairie (Microtus ochrogaster). and montane voles (M. montanus). differ in the distribution of oxytocin OT. and vasopressin AVP receptor binding in the brain. The present study examined the ontogenetic pattern of these receptor bindings in the lateral septum in both species to determine whether adult differences in the receptor binding are derived from a common pattern in development. In both species, OT and AVP receptor binding in the lateral septum were detected neonatally, increased during development, and reached the adult level at weaning third week. The progression of OT and AVP receptor differed, as OT receptor binding increased continually until weaning while AVP receptor binding did not change in the first week, increased rapidly in the second week, and was sustained thereafter. For both receptors, the binding increased more rapidly in montane than in prairie voles, resulting in species differences in receptor binding at weaning and in adulthood. Together, these data indicate that OT and AVP could affect the brain during development in a peptide- and species-specific manner in voles.

Evolution of the vasopressin/oxytocin superfamily: Characterization of a cDNA encoding a vasopressin-related precursor, preproconopressin, from the mollusc Lymnaea stagnalis
RE Van Kesteren, AB Smit, RW Dirksi, ND De With, WPM Geraerts, and J Joosse
Proc. Nadl. Acad. Sci. USA May 1992; 89: 4593-4597. Neurobiology

Although the nonapeptide hormones vasopressin, oxytocin, and related peptides from vertebrates and some nonapeptides from invertebrates share similarities in amino acid sequence, their evolutionary relationships are not dear. To investigate this issue, we doned a cDNA encoding a vasopressin-related peptide, Lys-conopressin, produced in the central nervous system of the gastropod mollusc Lymnaea stagnalis. The predicted preproconopressin has the overall architecture of vertebrate preprovasopressins, with a signal peptide, Lys-conopressin, that is flanked at the C terminus by an amidation signal and a pair of basic residues, followed by a neurophysin domain. The Lymnaea neurophysin and the vertebrate neurophysins share high sequence identity, which includes the conservation of all 14 cysteine residues. In addition, the Lymnaea neurophysin possesses unique structural characteristics. It contains a putative N-linked glycosylation site at a position in the vertebrate neurophysins where a strictly conserved tyrosine residue, which plays an essential role in binding of the nonapptide hormones, is found. The C-terminal copeptin homologous extension of the Lymnaea neurophysin has low sequence identity with the vertebrate counterparts and is probably not cleaved from the prohormone, as are the mammalin copeptins. The conopressin gene is expressed in only a few neurons in both pedal ganglia of the central nervous system. The conopressin transcript is present in two sizes, due to alternative use of polyadenylylation signals. The data presented here demonstrate that the typical organization of the prohormones of the vasopressin/oxytocin superfamily must have been present in the common ancestors of vertebrates and invertebrates.

A common allele in the oxytocin receptor gene (OXTR) impacts prosocial temperament and human hypothalamic-limbic structure and function
H Tosta, B Kolachanaa, S Hakimia, H Lemaitrea, BA Verchinskia, et al.
PNAS Aug 3, 2010; 107(31): 13936–13941

The evolutionarily highly conserved neuropeptide oxytocin is a key mediator of social and emotional behavior in mammals, including humans. A common variant (rs53576) in the oxytocin receptor gene (OXTR) has been implicated in social-behavioral phenotypes, such as maternal sensitivity and empathy, and with neuropsychiatric disorders associated with social impairment, but the intermediate neural mechanisms are unknown. Here, we used multimodal neuroimaging in a large sample of healthy human subjects to identify structural and functional alterations in OXTR risk allele carriers and their link to temperament. Activation and interregional coupling of the amygdala during the processing of emotionally salient social cues was significantly affected by genotype. In addition, evidence for structural alterations in key oxytocinergic regions emerged, particularly in the hypothalamus. These neural characteristics predicted lower levels of reward dependence, specifically in male risk allele carriers. Our findings identify sex-dependent mechanisms impacting the structure and function of hypothalamic-limbic circuits that are of potential clinical and translational significance.
Test of Association Between 10 SNPs in the Oxytocin Receptor Gene and Conduct Disorder
JT Sakai, TJ Crowley, MC Stallings, M McQueen, JK Hewitt, C Hopfer, et al.
Psychiatr Genet. 2012 Apr; 22(2): 99–102.

Animal and human studies have implicated oxytocin (OXT) in affiliative and prosocial behaviors. We tested whether genetic variation in the OXT receptor (OXTR) gene is associated with conduct disorder (CD).
Utilizing a family-based sample of adolescent probands recruited from an adolescent substance abuse treatment program, control probands and their families (total sample n=1,750), we conducted three tests of association with CD and 10 SNPs (single nucleotide polymorphisms) in the OXTR gene: (1) family-based comparison utilizing the entire sample; (2) within-Whites, case control comparison of adolescent patients with CD and controls without CD; and (3) within-Whites case-control comparison of parents of patients and parents of controls.
Family-based association tests failed to show significant results (no results p<0.05). While strictly correcting for the number of tests (α=0.002), adolescent patients with CD did not differ significantly from adolescent controls in genotype frequency for the OXTR SNPs tested; similarly, comparison of OXTR genotype frequencies for parents failed to differentiate patient and control family type, except a trend association for rs237889 (p=0.004). In this sample, 10 SNPs in the OXTR gene were not significantly associated with CD.

Leu55Pro transthyretin accelerates subunit exchange and leads to rapid formation of hybrid tetramers
CA Keetch, EHC Bromley, MG McCammon, N Wang, J Christodoulou, CV Robinson
JBC  Oct 11, 2005 M508753200.

Transthyretin is a tetrameric protein associated with the commonest form of

systemic amyloid disease. Using isotopically labeled proteins and mass spectrometry we compared subunit exchange in wild-type transthyretin with that of the variant associated with the most aggressive form of the disease, Leu55Pro. Wild-type subunit exchange occurs via both monomers and dimers , while exchange via dimers is the dominant mechanism for the Leu55Pro variant. Since patients with the Leu55Pro mutation are heterozygous, expressing both proteins simultaneously, we also analyzed the subunit exchange reaction between wild-type and Leu55Pro tetramers . We found that hybrid tetramers containing two or three Leu55Pro subunits dominate in the early stages of the reaction. Surprisingly we also found that in the presence of Leu55Pro transthyretin, the rate of dissociation of wild-type transthyretin is increased. This implies interactions between the two proteins that accelerate the formation of hybrid tetramers, a result with important implications for transthyretin amyloidos is.

Beyond Genetic Factors in Familial Amyloidotic Polyneuropathy: Protein Glycation and the Loss of Fibrinogen’s Chaperone Activity
G da Costa, RA Gomes, A Guerreiro, E Mateus, E Monteiro, et al.
PLoS ONE 2011; 6(10): e24850.

Familial amyloidotic polyneuropathy (FAP) is a systemic conformational disease characterized by extracellular amyloid fibril formation from plasma transthyretin (TTR). This is a crippling, fatal disease for which liver transplantation is the only effective therapy. More than 80 TTR point mutations are associated with amyloidotic diseases and the most widely accepted disease model relates TTR tetramer instability with TTR point mutations. However, this model fails to explain two observations. First, native TTR also forms amyloid in systemic senile amyloidosis, a geriatric disease. Second, age at disease onset varies by decades for patients bearing the same mutation and some mutation carrier individuals are asymptomatic throughout their lives. Hence, mutations only accelerate the process and non-genetic factors must play a key role in the molecular mechanisms of disease. One of these factors is protein glycation, previously associated with conformational diseases like Alzheimer’s and Parkinson’s. The glycation hypothesis in FAP is supported by our previous discovery of methylglyoxal-derived glycation of amyloid fibrils in FAP patients. Here we show that plasma proteins are differentially glycated by methylglyoxal in FAP patients and that fibrinogen is the main glycation target. Moreover, we also found that fibrinogen interacts with TTR in plasma. Fibrinogen has chaperone activity which is compromised upon glycation by methylglyoxal. Hence, we propose that methylglyoxal glycation hampers the chaperone activity of fibrinogen, rendering TTR more prone to aggregation, amyloid formation and ultimately, disease.

Aromatic Sulfonyl Fluorides Covalently Kinetically Stabilize Transthyretin to Prevent Amyloidogenesis while Affording a Fluorescent Conjugate
NP Grimster, S Connelly, A Baranczak, J Dong, …, JW Kelly
J Am Chem Soc. 2013 Apr 17; 135(15): 5656–5668.

Molecules that bind selectively to a given protein and then undergo a rapid chemoselective reaction to form a covalent conjugate have utility in drug development. Herein a library of 1,3,4-oxadiazoles substituted at the 2 position with an aryl sulfonyl fluoride and at the 5 position with a substituted aryl known to have high affinity for the inner thyroxine binding subsite of transthyretin (TTR) were conceived of by structure-based design principles and were chemically synthesized. When bound in the thyroxine binding site, most of the aryl sulfonyl fluorides react rapidly and chemoselectively with the pKa-perturbed K15 residue, kinetically stabilizing TTR and thus preventing amyloid fibril formation, known to cause polyneuropathy. Conjugation t50s range from 1 to 4 min, ~ 1400 times faster than the hydrolysis reaction outside the thyroxine binding site. Xray crystallography confirms the anticipated binding orientation and sheds light on the sulfonyl fluoride activation leading to the sulfonamide linkage to TTR. A few of the aryl sulfonyl fluorides efficiently form conjugates with TTR in plasma. A few of the TTR covalent kinetic stabilizers synthesized exhibit fluorescence upon conjugation and therefore could have imaging applications as a consequence of the environment sensitive fluorescence of the chromophore.

Identification of S-sulfonation and S-thiolation of a novel transthyretin Phe33Cys variant from a patient diagnosed with familial transthyretin amyloidosis
A Lim, T Prokaeva, ME Mccomb, LH Connors, M Skinner, and CE Costello
Protein Science 2003; 12:1775–1786.

Familial transthyretin amyloidosis (ATTR) is an autosomal dominant disorder associated with a variant form of the plasma carrier protein transthyretin (TTR). Amyloid fibrils consisting of variant TTR, wild-type TTR, and TTR fragments deposit in tissues and organs. The diagnosis of ATTR relies on the identification of pathologic TTR variants in plasma of symptomatic individuals who have biopsy proven amyloid disease. Previously, we have developed a mass spectrometry-based approach, in combination with direct DNA sequence analysis, to fully identify TTR variants. Our methodology uses immunoprecipitation to isolate TTR from serum, and electrospray ionization and matrix-assisted laser desorption/ionization mass spectrometry (MS) peptide mapping to identify TTR variants and posttranslational modifications. Unambiguous identification of the amino acid substitution is performed using tandem MS (MS/MS) analysis and confirmed by direct DNA sequence analysis. The MS and MS/MS analyses also yield information about posttranslational modifications. Using this approach, we have recently identified a novel pathologic TTR variant. This variant has an amino acid substitution (Phe — Cys) at position 33. In addition, like the Cys10 present in the wild type and in this variant, the Cys33 residue was both S-sulfonated and S-thiolated (conjugated to cysteine, cysteinylglycine, and glutathione). These adducts may play a role in the TTR fibrillogenesis.

Evolutionary relationships of lactate dehydrogenases (LDHs) from mammals, birds, an amphibian, fish, barley, and bacteria: LDH cDNA sequences from Xenopus, pig, and rat
S Tsuji, MA Qureshi, EW Hou, WM Fitch, and S S.-L. Li
Proc. Natl. Acad. Sci. USA Sep 1994; 91: 9392-9396. Evolution

The nucleotide sequences of the cDNAs encoding LDH (EC subunits LDH-A (muscle), LDH-B (liver), and LDH-C (oocyte) from Xenopus laevis, LDH-A (muscle) and LDH-B (heart) from pig, and LDH-B (heart) and LDH-C (testis) from rat were determined. These seven newly deduced amino acid sequences and 22 other published LDH sequences, and three unpublished fish LDH-A sequences kindly provided by G. N. Somero and D. A. Powers, were used to construct the most parsimonious phylogenetic tree of these 32 LDH subunits from mammals, birds, an amphibian, fish, barley, and bacteria. There have been at least six LDH gene duplications among the vertebrates. The Xenopus LDH-A, LDH-B, and LDH-C subunits are most closely related to each other and then are more closely related to vertebrate LDH-B than LDH-A. Three fish LDH-As, as well as a single LDH of lamprey, also seem to be more related to vertebrate LDH-B than to land vertebrate LDH-A. The mammalian LDH-C (testis) subunit appears to have diverged very early, prior to the divergence of vertebrate LDH-A and LDH-B subunits, as reported previously.

Evidence for neutral and selective processes in the recruitment of enzyme-crystallins in avian lenses
Graeme Wistow, Andrea Anderson, and Joram Piatigorsky
Proc. Natl. Acad. Sci. USA Aug 1990; 87: 6277-6280, Evolution

In apparent contrast to most other tissues, the ocular lenses in vertebrates show striking differences in protein composition between taxa, most notably in the recruitment of different enzymes as major structural proteins. This variability appears to be the result of at least partially neutral evolutionary processes, although there is also evidence for selective modification in molecular structure. Here we describe a bird, the chimney swift (Chaetura pelagica), that lacks δ-crystallin/ argininosuccinate lyase, usually the major crystallin of avian lenses. Clearly, δ-crystallin is not specifically required for a functionally effective avian lens. Furthermore the lens composition of the swift is more similar to that of the related hummingbirds than to that of the barn swallow (Hirundo rustica), suggesting that phylogeny is more important than environmental selection in the recruitment of crystallins. However differences in ε-crystallin/lactate dehydrogenase-B sequence between swift and hummingbird and other avian and reptilian species suggest that selective pressures may also be working at the molecular level. These differences also confirm the close relationship between swifts and hummingbirds.

Enzyme/crystallins and extremely high pyridine nucleotide levels in the eye lens.
Zigler, J. S., Jr.; Rao, P. V.
FASEB J. 1991; 3: 223-225.

Taxon-specific crystallins are proteins present in high abundance in the lens of phylogenetically restricted groups of animals. Recently it has been found that these proteins are actually enzymes which the lens has apparently adopted to serve as structural proteins. Most of these proteins have been shown to be identical to, or related to, oxidoreductases. In guinea pig lens, which contains zeta-crystallin, a protein with an NADPH dependent oxidoreductase activity, the levels of both NADPH and NADP* are extremely high and correlate with the concentration of zeta-crystallin. We report here nucleotide assays on lenses from vertebrates containing other enzyme/crystallins. In each case where the enzyme/crystallin is a pyridine nucleotide-binding protein the level of that particular nucleotide is extremely high in the lens. The presence of an enzyme/crystallin does not affect the lenticular concentrations of those nucleotides which are not specifically bound. The possibility that nucleotide binding may be a factor in the selection of some enzymes to serve as enzyme/crystallins is considered.

Comparison of stability properties of lactate dehydrogenase B4/ε-crystallin from different species
CEM Voorter, LTM Wintjes, PWH Heinstra, H Bloemendal and WW De Jong
Eur. J. Biochem. 1993; 211: 643-648

ε-Crystallin occurs as an abundant lens protein in many birds and in crocodiles and has been identified as heart-type lactate dehydrogenase (LDH-B4). Lens proteins have, due to their longevity and environmental conditions, extraordinary requirements for structural stability. To study lens protein stability, we compared various parameters of LDH-B4/ε-crystallin from lens and/or heart of duck, which has abundant amounts of this enzyme in its lenses, and of chicken and pig, which have no λ-crystallin. Measuring the thermostability of LDH-B4 from the different sources, the t50 values (temperature at which 50% of the enzyme activity remains after a 20-min period) for LDH-B4 from duck heart, duck lens and chicken heart were all found to be around 76°C whereas pig heart LDHB4 was less thermostable, having a t50 value of 625°C. A similar tendency was found with urea inactivation studies. Plotting the first-order rate constants obtained from inactivation kinetic plots against urea concentration, it was clear that LDH-B4 from pig heart was less stable in urea than the homologous enzymes from duck heart, chicken heart and duck lens. The duck and chicken enzymes were also much more resistant against proteolysis than the porcine enzyme. Therefore, it is concluded that avian LDH-B4 is structurally more stable than the homologous enzyme in mammals. This greater stability might make it suitable to function as a ε-crystallin, as in duck, but is not necessarily associated with high lens expression, as in chicken.

Duck lens ε-crystallin and lactate dehydrogenase B4 are identical: A single-copy gene product with two distinct functions
W Hendriks, JWM Mulders, MA Bibby, C Slingsby, H Bloemendal, and WW De Jong
Proc. Natl. Acad. Sci. USA Oct 1988; 85: 7114-7118. Biochemistry

To investigate whether or not duck lens ε-crystaliin and duck heart lactate dehydrogenase (LDH) B4 are the product of the same gene, we have isolated and sequenced cDNA clones of duck ε-crystallin. By using these clones we demonstrate that there is a single-copy Ldh-B gene in duck and in chicken. In the duck lens this gene is overexpressed, and its product is subject to posttranslational modification. Reconstruction of the evolutionary history of the LDH protein family reveals that the mammalian Ldh-C gene most probably originated from an ancestral Ldh-A gene and that the amino acid replacement rate in LDH-C is approximately 4 times the rate in LDH-A. Molecular modeling of LDH-B sequences shows that the increased thermostability of the avian tetramer might be explained by mutations that increase the number of ion pairs. Furthermore, the replacement of bulky side chains by glycines on the corners of the duck protein suggests an adaptation to facilitate close packing in the lens.

Lactate Dehydrogenase A as a Highly Abundant Eye Lens Protein in Platypus (Ornithorhynchus anatinus): Upsilon (υ)-Crystallin
T van Rheede,  R Amons, N Stewart, and WW de Jong
Mol. Biol. Evol. 2003; 20(06):994–998.

Vertebrate eye lenses mostly contain two abundant types of proteins, the α-crystallins and the β/λ-crystallins. In addition, certain housekeeping enzymes are highly expressed as crystallins in various taxa. We now observed an unusual approximately 41-kd protein that makes up 16% to 18% of the total protein in the platypus eye lens. Its cDNA sequence was determined, which identified the protein as muscle-type lactate dehydrogenase A (LDH-A). It is the first observation of LDH-A as a crystallin, and we designate it upsilon (υ)-crystallin. Interestingly, the related heart-type LDH-B occurs as an abundant lens protein, known as ε-crystallin, in many birds and crocodiles. Thus, two members of the ldh gene family have independently been recruited as crystallins in different higher vertebrate lineages, suggesting that they are particularly suited for this purpose in terms of gene regulatory or protein structural properties. To establish whether platypus LDH-A/υ-crystallin has been under different selective constraints as compared with other vertebrate LDH-A sequences, we reconstructed the vertebrate Ldh-A gene phylogeny. No conspicuous rate deviations or amino acid replacements were observed.

Isozymes, moonlighting proteins and promiscous enzymes
M Nath Gupta, M Kapoor, AB Majumder and V Singh
Current Science Apr 2011; 100(8): 1152-1162.

The structures of isoenzymes differ and yet these catalyse the same type of reaction. These structures evolved to suit the physiological needs and are located in different parts of cells or tissues. Moonlighting proteins represent the same structure performing very different biological functions. Biological promiscuity reveals that the same active sites can catalyse different types of reactions. These three different phenomena, all illustrate similar evolutionary strategies. Viewed together, it emerges that biologists need to take a hard look at the ‘structure–function’ paradigm as well as the notions of biological specificity. Meanwhile, biotechnologists  continue to exploit the opportunities which ‘nonspecificity’ offers.

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