Larry H. Bernstein, MD, FCAP, Author and Curator
Leaders in Pharmaceutical Innovation
Introduction
This contribution to a series of articles on the history of physiology, biochemistry, molecular biology, and medicine, examines several instances of conserved proteins over perhaps millions of years and across species of eukaryotes. These findings appear to be unique in some respects within certain specific areas of scientific investigation, but while the accuracy of this observation might perhaps be open to challenge, the clarification of these observations should open up and enrich our understanding of biological function.
Conversations with Jose Eduardo des Salles Rosalino raised this question? How is it that developments late in the 20th century diverted the attention of biological processes from a dynamic construct involving interacting chemical reactions under rapidly changing external conditions effecting tissues and cell function to a rigid construct that is determined unilaterally by the genome construct, diverting attention from mechanisms essential for seeing the complete cellular construct?
He calls attention to the article titled Neo -Darwinism, The Modern Synthesis and Selfish Genes that bares no relationship with Physiology with Molecular Biology J. Physiol 2011; 589(5): 1007-11 by Denis Noble. Further, he identifies it as the key factor required in order to understand the dislodgement of physiology as the foundation of medical reasoning. This is because of the near unilateral emphasis of genomic activity as a determinant of cellular. The DNA to protein link goes from triplet sequence to amino acid sequence. That is the realm of genetics. However, protein conformation, activity and function requires that environmental and micro-environmental factors should be considered (Biochemistry). If that were not the case, we have no way to bridge the gap between the genetic code and the evolution of cells, tissues, organs, and organisms.
He continues with this example. I would like to stress in the cAMP coupled hormonal response, the transfer of conformation from protein to protein is paramount. For instance, if your scheme goes beyond cAMP, it will show an effect over a self-assembly (inhibitor protein and protein kinase). This effect is not in any way determined by the translation of the genetic code. It is an energetic homeostatic response. Most important, sequence alone does not explain conformation, activity and function of regulatory proteins. If this important mechanism was not ignored, the work of S Prusiner would be easily understood. For, self-assembly versus change in covalent modification of proteins (see R. A Kahn and A. G Gilman 1984 J. Biol. Chem. v. 259,n 10 pp6235-6240.) In this case, trimeric or dimeric G does not matter.” Signaling transduction tutorial”.
We know that proteins have primary, secondary, and tertiary structure. The primary structure is transcribed from the genetic code. The secondary structure constitutes the first level of protein folding. It is stabilized by strong interactions between side chains of neighboring amino acids, and it consists of the linear sequence of amino acids connected by peptide linkages. Altering a single amino acid is associated with a gene mutation, which might result in a non-functioning protein. The secondary structure is primarily responsible for lining up the active site residues within a protein structure, and it is stabilized by a few, but very strong disulfide bonds between adjacent cysteine amino acids within the polypeptide chains.
The coiling or folding of a polypeptide chain gives the protein its 3-D shape. The alpha (α) helix structure resembles a coiled spring and is secured by hydrogen bonding in the polypeptide chain. The beta (β) pleated sheet is a structure that appears to be folded or pleated and is held together by hydrogen bonding between polypeptide units of the folded chain that lie adjacent to one another. The 3-D structure of the polypeptide chain of a protein is determined by several types of bonds and forces that hold a protein in its tertiary structure. Hydrophobic interactions greatly contribute to the folding and shaping of a protein. The “R” group of the amino acid is either hydrophobic or hydrophilic, which in effect either causes the “R” to avoid water and pull into the center of the molecule, or alternatively, to seek contact with water. Due to protein folding, ionic bonding can occur between the positively and negatively charged “R” groups that come in close contact with one another. Folding can also result in covalent bonding between the “R” groups of cysteine amino acids. This type of bonding forms what is called a disulfide bridge. Interactions called van der Waals forces also assist in the stabilization of protein structure. These interactions pertain to the attractive and repulsive forces that occur between molecules that become polarized. These forces contribute to the bonding that occurs between molecules.
Quaternary structure refers to the structure of a protein macromolecule formed by interactions between multiple polypeptide chains. Each polypeptide chain is referred to as a subunit. Proteins with quaternary structure may consist of more than one of the same or different type of protein subunit. This is common for catalytical enzymes and for plasma circulating transport proteins. It is also consequential for physiological and pathophysiological events. Typical examples are hemoglobin and transthyretin.
Self-assembly is a phenomenon where the components of a system assemble themselves spontaneously via an interaction to form a larger functional unit. This spontaneous organization can be due to direct specific interaction and/or indirectly through their environment. The spatial arrangements of these self-assembled molecules can be potentially used to build increasingly complex structures. Some properties of atoms and molecules enable them to arrange themselves into patterns.
Self-assembly is an equilibrium process where the assembled components are in equilibrium with the individual components. Self-assembly is driven by the minimization of Gibbs free energy. The minimization of Gibbs free energy is attained by the minimization of repulsive and the maximization of attractive molecular interactions. In addition, the lower free energy is usually a result of a weaker intermolecular force between self-assembled moieties and is essentially enthalpic in nature.
The thermodynamics of the self-assembly process can be represented by a simple Gibbs free energy equation:
\Delta G_{SA} = \Delta H_{SA} -T \Delta S_{SA} \,
where if \Delta G_{SA} \, is negative, self-assembly is a spontaneous process. \Delta H_{SA} \, is the enthalpy change of the process and is largely determined by the potential energy/intermolecular forces between the assembling entities. \Delta S_{SA} \, is the change in entropy associated with the formation of the ordered arrangement. In general, the organization is accompanied by a decrease in entropy and in order for the assembly to be spontaneous the enthalpy term must be negative and in excess of the entropy term. This equation shows that as the value of T \Delta S_{SA} \, approaches the value of \Delta H_{SA} \, and above a critical temperature, the self-assembly process will become progressively less likely to occur and spontaneous self-assembly will not happen.
The self-assembly is governed by the normal processes of nucleation and growth. Small assemblies are formed because of their increased lifetime as the attractive interactions between the components lower the Gibbs free energy. As the assembly grows, the Gibbs free energy continues to decrease until the assembly becomes stable enough to last for a long period of time. The necessity of the self-assembly to be an equilibrium process is defined by the organization of the structure which requires non-ideal arrangements to be formed before the lowest energy configuration is found.
Intermolecular forces govern the particle interaction in self-assembled systems. The forces tend to be intermolecular in type rather than ionic or covalent because ionic or covalent bonds will “lock” the assembly into non-equilibrium structures. The types intermolecular forces seen in self-assembly processes are van der Waals, hydrogen bonds, and weak polar forces, just to name a few. In self-assembly, regular structural arrangements are frequently observed, therefore there must be a balance of attractive and repulsive between molecules otherwise an equilibrium distance will not exist between the particles. The repulsive forces can be electron cloud-electron cloud overlap or electrostatic repulsion. (Wikipedia)
This description fits nicely into the intermolecular interactions between protein polypeptides in both tertiary and quaternary structure. This principle is being utilized in the engineering of nanoparticles that will self-assemble for one or both of two reasons: molecular interactions and external direction. The assembly of nanoparticles of two different materials into a binary nanoparticle superlattice is a promising way of synthesizing a large variety of materials
Prions , Stanley B. Prusiner (PNAS 1998, Nov 10; 95(23): 13363–13383, http://dx.doi.org:/10.1073/pnas.95.23.13363
Prion diseases may present as genetic, infectious, or sporadic disorders, all of which involve modification of the prion protein (PrP). Prions are transmissible particles that are devoid of nucleic acid and seem to be composed exclusively of a modified protein (PrPSc). The normal, cellular PrP (PrPC) is converted into PrPSc through a posttranslational process during which it acquires a high β-sheet content. The species of a particular prion is encoded by the sequence of the chromosomal PrP gene of the mammals in which it last replicated. In contrast to pathogens carrying a nucleic acid genome, prions appear to encipher strain-specific properties in the tertiary structure of PrPSc. Transgenetic studies argue that PrPSc acts as a template upon which PrPC is refolded into a nascent PrPSc molecule through a process facilitated by another protein.
Miniprions generated in transgenic mice expressing PrP, in which nearly half of the residues were deleted, exhibit unique biological properties and should facilitate structural studies of PrPSc. Knowledge about prions has profound implications for studies of the structural plasticity of proteins.
Prusiner relates:
Libyan Jews living in Israel developed CJD about 30 times more frequently than other Israelis. This finding prompted some investigators to propose that the Libyan Jews had contracted CJD by eating lightly cooked brain from scrapie-infected sheep when they lived in Tripoli prior to emigration. Subsequently, the Libyan Jewish patients were all found to carry a mutation at codon 200 in their prion protein (PrP) gene.
I was beginning a residency in neurology in 1972 and was most impressed by a disease process that could kill my patient in 2 months by destroying her brain while her body remained unaffected by this process. No febrile response, no leukocytosis or pleocytosis, no humoral immune response, and yet I was told that she was infected with a “slow virus.” In scrapie, kuru, CJD, and all of the other disorders now referred to as prion diseases, spongiform degeneration and astrocytic gliosis is found upon microscopic examination of the CNS. The extent of reactive gliosis correlates with the degree of neuron loss.
Neuropathologic changes in Swiss mice after inoculation with RML scrapie prions.
In view of daunting logistical problems, the identification of an inoculum that produced scrapie in the golden Syrian hamster (SHa) in ≈70 days after intracerebral inoculation proved to be an important advance once an incubation time assay was developed. It provided a means to assess quantitatively those fractions that were enriched for infectivity and those that were not. A protocol was developed for separating scrapie infectivity from most proteins and nucleic acids. With a ≈100-fold purification of infectivity relative to protein, >98% of the proteins and polynucleotides were eliminated, permitting more reliable probing of the constituents of these enriched fractions.
A particular macromolecule was required for infectivity and this macromolecule was a protein. The experimental findings extended earlier observations on resistance of scrapie infectivity to UV irradiation at 250 nm in that the four different procedures used to probe for a nucleic acid are based on physical principles that are independent of UV radiation damage. I defined prions as proteinaceous infectious particles that resist inactivation by procedures that modify nucleic acids.The best current working definition of a prion is a proteinaceous infectious particle that lacks nucleic acid, because a wealth of data supports the contention that scrapie prions are composed entirely of a protein that adopts an abnormal conformation. From a broader perspective, prions are elements that impart and propagate conformational variability.
JEDS continues with his observations about the uniqueness of the functional understanding of proteins. He refers to lessons derived from discussions with Leloir, and an imaginary dissertation by Schrodinger to Darlington and Haldane, the two distinguished biologists of their time. His vision was that that biochemistry could provide two legs to support the advance of biology and medicine (one leg was for normal physiology and the other the solitary leg of molecular biology, for genetics). This view includes a chemical reactivity scenario that in Leloir´s vision on the subject, he considers to be of paramount importance. A chemical reaction point of view prevents any scientific supporting leg to be placed in an opposite or wrong position as it is in the simple statement that ALL information in life moves from DNA, through RNA and to proteins.
Complex life forms have followed what we usually understand as evolution, variation and selection leading to new forms of life and old ones being extinct without a direct selective pressure upon its amount of DNA. For the more complex life forms, neutral mutations would be very important in the evolution process than for bacteria. However, it also, may shed some new light upon the controversy of a small step in evolution as Charles Darwin proposed, and in addition, some more modern views that have indicated that abrupt changes might have occurred during the biological time line. Neutral evolution might have happened as small step evolution, as imagined by Darwin. Only after a rather long period of time, and after a large series of neutral mutations, a new function, or a new phenotype appears after a last small step, the last neutral mutation.
A set of genes from both parents seems to be closer to the common ancestry of both parents than to either parent. These ancestral genes that are present may be expressed in complex living beings, are no longer present in modern microorganisms genomes as well as previous non-coding DNA. This last aspect of the genome makes clear the difference in evolution pathways of microorganisms in comparison with more complex forms of life.
Crystallins of the eye lens, cornea, and retina
Regulation of gene expression by Pax6 in ocular cells: a case of tissue-preferred expression of crystallins in lens
A Cvekl, Y Yang, BK Chauhan and K Cveklova
Int. J. Dev. Biol. 2004; 48: 829-844. http://dx.doi.org:/10.1387/ijdb.041866ac
Lens development is an excellent model for genetic and biochemical studies of embryonic induction, cell cycle regulation, cellular differentiation and signal transduction. Differentiation of lens is characterized by lens-preferred expression and accumulation of water-soluble proteins, crystallins. Crystallins are required for light transparency, refraction and maintenance of lens integrity. Here, we review mechanisms of lens-preferred expression of crystallin genes by employing synergism between developmentally regulated DNA-binding transcription factors: Pax6, c-Maf, MafA/L-Maf, MafB, NRL, Sox2, Sox1, RARβ/RXRβ, RORα, Prox1, Six3, γFBP-B and HSF2. These factors are differentially expressed in lens precursor cells, lens epithelium and primary and secondary lens fibers. They exert their function in combination with ubiquitously expressed factors (e.g. AP-1, CREB, pRb, TFIID and USF) and co-activators/chromatin remodeling proteins (e.g. ASC-2 and CBP/p300). A special function belongs to Pax6, a paired domain and homeodomain-containing protein, which is essential for lens formation. Pax6 is expressed in lens progenitor cells before the onset of crystallin expression and it serves as an important regulatory factor required for expression of c-Maf, MafA/L-Maf, Six3, Prox1 and retinoic acid signaling both in lens precursor cells and the developing lens. The roles of these factors are illustrated by promoter studies of mouse αA-, αB-, γF- and guinea pig ζ-crystallins. Pax6 forms functional complexes with a number of transcription factors including the retinoblastoma protein, pRb, MafA, Mitf and Sox2. We present novel data showing that pRb antagonizes Pax6-mediated activation of the αA-crystallin promoter likely by inhibiting binding of Pax6 to DNA.
The transcription factor D-Pax2 regulates Crystallin production during eye development in Drosophila melanogaster
K Dziedzic, J Heaphy, H Prescott, and J Kavaler
Dev Dyn. 2009 Oct; 238(10): 2530–2539. http://dx.doi.org:/10.1002/dvdy.22082
The generation of a functioning Drosophila eye requires the coordinated differentiation of multiple cell types and the morphogenesis of eye-specific structures. Here we show that D-Pax2 plays a significant role in lens development through regulation of the Crystallin gene and that Crystallin is also expressed in D-Pax2+ cells in the external sensory organs. Loss of D-Pax2 function leads to loss of Crystallin expression in both eyes and bristles. A 2.3 kilobase upstream region of the Crystallin gene can drive GFP expression in the eye and is dependent on D-Pax2. In addition, D-Pax2 binds to an evolutionarily conserved site in this region which, by itself, is sufficient to drive GFP expression in the eye. However, mutation of this site does not greatly affect the regulatory region’s function. The data indicate that D-Pax2 acts to promote lens development by controlling the production of the major protein component of the lens. Whether this control is direct or indirect remains unresolved.
Cubozoan jellyfish: an Evo/Devo model for eyes and other sensory systems
J PIATIGORSKY, and Z KOZMIK
Int. J. Dev. Biol. 2004; 48: 719-729. http://dx.doi.org:/10.1387/ijdb.041851jp
Cnidaria are the most basal phylum containing a well-developed visual system located on specialized sensory structures (rhopalia) with eyes and statocyts. We have been exploring the cubozoan jellyfish, Tripedalia cystophora. In addition to containing simple photoreceptive ocelli, each rhopalium in Tridedalia has a large and small complex, camera-type eye with a cellular lens containing three distinct families of crystallins which apparently serve non-lenticular functions. Thus, Tridpedalia recruited crystallins by a gene sharing strategy as have mollusks and vertebrates. Tripedalia has a single Pax gene, PaxB, which encodes a structural and functional Pax 2/5/8-like paired domain as well as an octapeptide and Pax6-like homeodomain. PaxB binds to and activates Tripedalia crystallin promoters (especially J3-crystallin) and the Drosophila rhodopsin rh6 gene in transfection tests and induces ectopic eyes in Drosophila. In situ hybridization showed that PaxB and crystallin genes are expressed in the lens, retina and statocysts. We suggest from these results that an ancestral PaxB gene was a primordial gene in eye evolution and that eyes and ears (mechanoreceptors) may have had a common evolutionary origin. Thus, the numerous structural and molecular features of Tridpalia rhopalia indicate that ancient cubozoan jellyfish are fascinating models for evo/devo insights into eyes and other sensory systems.
aB-crystallin, a low-molecular-weight heat shock protein, acts as a regulator of platelet function
O. Kozawa, H. Matsuno, M. Niwa, D. Hatakeyama, K. Kato, and T. Uematsu
Cell Stress & Chaperones 2001; 6 (1): 21–28
It has recently been reported that aB-crystallin, a low-molecular-weight heat shock protein, may be released from cells by mechanical stretch. We investigated a physiological role of aB-crystallin in platelet function. aB-crystallin inhibited platelet aggregation induced by thrombin or botrocetin in hamsters and humans. These platelets had specific binding sites for aB-crystallin. Moreover, aB-crystallin significantly reduced thrombin-induced Ca21 influx and phosphoinositide hydrolysis by phospholipase C in human platelets. Additionally, plasma levels of aB-crystallin were markedly elevated in cardiomyopathic hamsters. Levels of aB-crystallin in vessel walls after endothelial injury were markedly reduced. Therefore, our results suggest that aB-crystallin, which is discharged from vessel walls in response to endothelial injury, acts intercellularly as a regulator of platelet function.
The role of Eph receptors in lens function and disease
AI SON, JE PARK, and RP ZHOU
Sci China Life Sci. 2012 May ; 55(5): 434–443.
http://dx.doi.org:/10.1007/s11427-012-4318-7.
In recent years the Eph family of receptor tyrosine kinases has been identified as a key regulator in lens clarity. In this review we discuss the roles of the Eph receptors in lens biology and cataract development.
As the primary role of the lens is to provide proper light refraction to the retina, the organ must possess several inherent characteristics, including stability, clarity, and the proper refractive index, to function appropriately. This is made possible in part through key features of the cytoplasmic content of lens fiber cells. One particularly unique aspect of lens fiber cells is their absence of organelles. Occurring during the later aspects of the differentiation process, fiber cells experience a gradual degradation of nuclei and organelles over a span of several days in what has been described as an “attenuated apoptosis”. This event is crucial for maintaining lens transparency as the absence of internal cellular structures allows light to travel unimpeded through the structure to the retina.
In addition to the loss of organelles, fiber cells achieve their appropriate refractive properties through regulation of their protein content. The bulk of the lens consists of soluble proteins known as the crystallins, a family comprising of α-, β-, and γ-crystallins. Crystallins represent 90% of the protein in the lens and provide the proper refractive medium for light passage within the lens. In addition to structural functions, α-crystallin is capable of chaperone activity, preventing aggregation of proteins denatured by various stressors. This chaperone activity is particularly important in the prevention of protein aggregation that may otherwise lead to insolubility and result in cataract formation. α-crystallins are expressed in both the lens epithelium and fiber cells, while β- and γ-crystallins are exclusively in differentiated lens fiber cells and are indicators of lens cell differentiation.
Lens clarity depends on the proper regulation of several critical features including structural integrity, cellular adhesion and packing, protein stability, cell-cell communication, and circulation. Mutations in the Eph receptor EPHA2 have been recently shown to cause cataracts in humans. The loss of functions of Eph receptors may affect one or several of these processes resulting in cataractogenesis.
The protein structure is highly conserved between both the EphA and EphB subgroups. The extracellular portion of the receptors contains an ephrin ligand-binding domain at the N-terminus, followed by a cystein-rich region epidermal growth factor (EGF) repeat motif and two fibronectin type-III repeats. The intercellular portion contains the signaling components which include a juxtamembrane region, a tyrosine kinase domain, a sterile alpha motif (SAM), and a PDZ binding domain at the end of the C-terminus.
Thus far, only EphA2 and ephrin-A5 have been found to have major roles in lens development and function. Expression of EphA2 in the lens has been reported to be within the fiber layer, most notably within the subcortical region, with some expression in epithelial cell layers. Ephrin-A5 has similar localizations, with expression observed in the subcortical fiber layer as well as within the lens epithelial layer. In the mature lens, both EphA2 and ephrin-A5 expression has been observed in the short edges of lens fiber cells when lenses are oriented in cross-section.
,u-Crystallin is a mammalian homologue of Agrobacterium ornithine cyclodeaminase and is expressed in human retina
RY Kim, R Gassert, and GJ Wistow
Proc. Natl. Acad. Sci. USA 1992 Oct; 89: 9292-9296. Evolution.
µ-Crystallin is the major component of the eye lens in several Australian marsupials. The complete sequence of kangaroo -crystalln has now been obtained by cDNA cloning. The predicted amino acid sequence shows similarity with ornithine cyclodeaminases encoded by the tumor-inducing (Ti) plasmids of Agrobacterium tumefaciens. Until now, neither ornithine cyclodeaminase nor any structurally related enzymes have been observed in eukaryotes. RNA analysis of kangaroo tissues shows that µ-crystallin is expressed at high abundance in lens, but outside the lens µ-crystallin is preferentially expressed in neural tissues, retina, and brain. An
almost full-length cDNA for µ-crystallin was cloned from human retina. In human tissues, µ-crystallin mRNA is present in neural tissue, muscle, and kidney. This pattern of expression and relationship to an enzyme involved in unusual amino acid metabolism suggests the interesting possibility that mammalian µ-crystallins could be enzymes participating in processes such as osmoregulation or the metabolism of excitatory amino acids.
Ocular Aldehyde Dehydrogenases: Protection against Ultraviolet Damage and Maintenance of Transparency for Vision
Y Chen, DC Thompson, V Koppaka, JV Jester, and V Vasiliou
Prog Retin Eye Res. 2013 Mar; 33: 28–39.
http://dx.doi.org:/10.1016/j.preteyeres.2012.10.001
Aldehyde dehydrogenase (ALDH) enzymes catalyze the NAD(P)+-dependent oxidation of a wide variety of endogenous and exogenous aldehydes to their corresponding acids. Some members of the ALDH superfamily of enzymes are abundantly expressed in the mammalian cornea and lens in a taxon-specific manner. Considered to be corneal and lens crystallins, they confer protective and transparent properties upon these ocular tissues. ALDH3A1 is highly expressed in the cornea of most mammals, with the exception of rabbit that expresses exclusively ALDH1A1 in the cornea. ALDH1A1 is present in both the cornea and lens of several animal species. As a result of their catalytic and non-catalytic functions, ALDH3A1 and ALDH1A1 proteins protect inner ocular tissues from ultraviolet radiation and reactive oxygen-induced damage. In addition, these corneal crystallins contribute to cellular transparency in corneal stromal keratocytes, supporting a structural role of these ALDH proteins. A putative regulatory function of ALDH3A1 on corneal cell proliferation has also been proposed. Finally, the three retinaldehye dehydrogenases cooperatively mediate retinoic acid signaling during the eye development.
Transthyretin
Protein aggregation: Mechanisms and functional consequences
G Invernizzi, E Papaleo, R Sabate, S Ventura
Int J Biochem & Cell Biol Sep 2012; 44(9): 1541-1554
Understanding the mechanisms underlying protein misfolding and aggregation has become a central issue in biology and medicine. Compelling evidence show that the formation of amyloid aggregates has a negative impact in cell function and is behind the most prevalent human degenerative disorders, including Alzheimer’s Parkinson’s and Huntington’s diseases or type 2 diabetes. Surprisingly, the same type of macromolecular assembly is used for specialized functions by different organisms, from bacteria to human. Here we address the conformational properties of these aggregates, their formation pathways, their role in human diseases, their functional properties and how bioinformatics tools might be of help to study these protein assemblies.
The most employed three-dimensional representation for in silico studies of protein aggregation. (A) All-atom explicit solvent representation adopted in classical MD simulations. Transthyretin (TTR) is shown as an example in its dimeric form (pdb entry 1F41). The solvent molecule, Na+ and Cl− ions are shown as red sticks, blue and light blue spheres, respectively. The two polypeptide chains of TTR are shown as green and cyan cartoon, respectively. (B) Coarse grained model of TTR. In coarse grained calculations, the protein residues are represented by different pseudoatoms (beads) which should capture the essential properties of each residue side-chain.
http://www.sciencedirect.com/science/article/pii/S1357272512001896#fig0010 http://ars.els-cdn.com/content/image/1-s2.0-S1357272512001896-gr2.sml
Signaling by vitamin A and retinol-binding protein in regulation of insulin responses and lipid homeostasis
Daniel C. Berry, Noa Noy
Biochim Biophys Acta (BBA) – Molec Cell Biol of Lipids, Jan 2012; 1821(1): 168-176
► Holo-RBP, which transports vitamin A in blood, is a signaling molecule. ► STRA6 functions both as a vitamin A transporter and as a surface signaling receptor activated by holo-RBP. ► Activation of STRA6 by RBP–ROH triggers a JAK/STAT cascade, thereby inducing gene trascription. ► Some genes induced by RBP–ROH/STRA6/JAK/STAT signaling are involved in regulating insulin responses and lipid metabolism.
Binding of RBP–ROH to STRA6 induces the phosphorylation of a tyrosine residue in the receptor C-terminus, thereby activating a JAK/STAT signaling cascade. Consequently, in STRA6-expressing cells such as adipocytes, RBP–ROH induces the expression of STAT target genes, including SOCS3, which suppresses insulin signaling, and PPARγ, which enhances lipid accumulation. RBP–retinol thus joins the myriad of cytokines, growth factors and hormones which regulate gene transcription by activating cell surface receptors that signal through activation of Janus kinases and their associated transcription factors STATs.

STRA6 transduces RBP–retinol signaling to trigger a JAK.STAT cascade that regulates insulin responses and lipid homeostasis
STRA6 transduces RBP–retinol signaling to trigger a JAK/STAT cascade that regulates insulin responses and lipid homeostasis
http://ars.els-cdn.com/content/image/1-s2.0-S1388198111001211-gr1.sml
Residues that stabilize the interactions of RBP with TTR are highlighted in blue. The location of these residues emphasize that interactions of RBP with TTR block the entrance to the RBP ligand-binding pocket.
http://ars.els-cdn.com/content/image/1-s2.0-S1388198111001211-gr2.sml
The three dimensional crystal structure of the retinol–RBP–TTR complex. Human retinol–RBP–TTR [29] (PDB ID:1QAB) was generated using Pymol (http://www.pymol.org/). The TTR tetramer (magenta) is comprised of a dimer of dimers with two RBP molecules (red) bound to the opposite dimers. Interactions between RBP and TTR are mediated by residues at the entrance to the ligand binding pocket and span across the two TTR dimers.
http://ars.els-cdn.com/content/image/1-s2.0-S1388198111001211-gr3.sml
http://www.sciencedirect.com/science/article/pii/S1388198111001211#f0015
Fourier Transform Infrared Spectroscopy Provides a Fingerprint for the Tetramer and for the Aggregates of Transthyretin
Y Cordeiro, J Kraineva, MC Suarez, AG Tempesta, JW Kelly, JL Silva, et al.
Biophys J, 1 Aug 2006; 91(3): 957-967
Transthyretin (TTR) is an amyloidogenic protein whose aggregation is responsible for several familial amyloid diseases. Here, we use FTIR to describe the secondary structural changes that take place when wt TTR undergoes heat- or high-pressure-induced denaturation, as well as fibril formation. Upon thermal denaturation, TTR loses part of its intramolecular β-sheet structure followed by an increase in nonnative, probably antiparallel β-sheet contacts (bands at 1616 and 1686 cm−1) and in the light scattering, suggesting its aggregation. Pressure-induced denaturation studies show that even at very elevated pressures (12 kbar), TTR loses only part of its β-sheet structure, suggesting that pressure leads to a partially unfolded species. On comparing the FTIR spectrum of the TTR amyloid fibril produced at atmospheric pressure upon acidification (pH 4.4) with the one presented by the native tetramer, we find that the content of β-sheets does not change much upon fibrillization; however, the alignment of β-sheets is altered, resulting in the formation of distinct β-sheet contacts (band at 1625 cm−1). The random-coil content also decreases in going from tetramers to fibrils. This means that, although part of the tertiary- and secondary-structure content of the TTR monomers has to be lost before fibril formation, as previously suggested, there must be a subsequent reorganization of part of the random-coil structure into a well-organized structure compatible with the amyloid fibril, as well as a readjustment of the alignment of the β-sheets. Interestingly, the infrared spectrum of the protein recovered from a cycle of compression-decompression at pD 5, 37°C, is quite similar to that of fibrils produced at atmospheric pressure (pH 4.4), which suggests that high hydrostatic pressure converts the tetramers of TTR into an amyloidogenic conformation.
Multiple hepatocyte-enriched nuclear factors function in the regulation of transthyretin and alpha 1-antitrypsin genes
R H Costa, D R Grayson and J E Darnell Jr
PNAS Jun 1, 1988; 85(11): 3840-3844
Double-stranded oligodeoxynucleotides that represent protein binding sites in the regulatory regions of the mouse genes encoding transthyretin (TTR) and alpha 1-antitrypsin (alpha 1-AT) bound a nuclear protein factor(s) found mainly in hepatocytes. A site in the regulatory region of the gene encoding rat serum albumin and, surprisingly, a region in the simian virus 40 enhancer also bind the same factor. Oligodeoxynucleotide affinity chromatography (with one of the TTR binding sites) allowed a 500-fold purification of the protein. The purified protein protected similar portions of all the regulatory regions, as well as the simian virus 40 core C enhancer element, from digestion with DNase I. A DNA-binding protein previously purified from liver by virtue of its ability to bind to several virus enhancer sequences also binds to TTR, alpha 1-AT, and albumin regulatory sites. Thus, all these binding sites, which contain only minimal sequence similarity, may bind to a single protein, or a similar family of proteins, that activates liver-specific transcription of coordinately expressed genes.
Retinal Anatomy and Function of the Transthyretin Null Mouse
BV Buia, JA Armitagea, EL Fletchera, SJ Richardsonb, G Schreiberb and AJ Vingrysa. Exp. Eye Res. 2001; 73: 651-659
TTR null mice show no anomalies of receptoral (P3) nor post-receptoral (b-wave) ERG components compared with wild-type mice. although circulating plasma levels of retinol and retinol binding protein (RBP) are extremely low, this reduction has little effect on the retinal structure or function of the TTR null mouse. These data are consistent with the existence of mechanisms for the transport of retinol to the retina independent of the classical retinol-RBP-TTR complex.
Post-translational modifications of transthyretin affect the triiodonine-binding potential
A Henze, T Homann, M Serteser, O Can, O Sezgin, et al.
J. Cell. Mol. Med. 2014; XX(X): 1-12
This study provides evidence that oxidative modifications of Cys10 seem to affect binding of T3 to TTR probably because of the introduction of a sterical hindrance and induction of conformational changes. As oxidative modifications can be dynamically regulated, this may represent a sensitive mechanism to adjust thyroid hormone availability.
Plasma and cellular retinoid-binding proteins and transthyretin are all localized in the islets of Langerhans in the rat
M Kato, K Kato, WS Blaner, BS Chertow, and DS Goodman
Proc. Nati. Acad. Sci. 1985; 82: 2488-2492
Unusually high levels of CRBP, RBP, TTR, and cellular retinoic acid BP were found in rat islets. The findings suggest that retinoids and their binding proteins may play important metabolic roles within islet cells, and hence that they may be involved in some way in the biological, endocrine function of the islets.
Potent Kinetic Stabilizers that Prevent Transthyretin-mediated Cardiomyocyte Proteotoxicity
MM Alhamadsheh, S Connelly, A Cho, N Reixach,..JW Kelly, and IA Graef
Sci Transl Med. 2011 Aug 24; 3(97): 97ra81.
http://dx.doi.org:/10.1126/scitranslmed.3002473
The V122I mutation that alters the stability of transthyretin (TTR) affects 3–4% of African Americans and leads to amyloidogenesis and development of cardiomyopathy. In addition, 10–15% of individuals over the age of 65 develop senile systemic amyloidosis (SSA) and cardiac TTR deposits due to wild-type TTR amyloidogenesis. We developed a fluorescence polarization-based HTS screen, which identified several new chemical scaffolds targeting TTR. These novel compounds were potent kinetic stabilizers of TTR and prevented tetramer dissociation, unfolding and aggregation of both wild type and the most common cardiomyopathy-associated TTR mutant, V122I-TTR. These compounds effectively inhibited the proteotoxicity of V122I-TTR towards human cardiomyocytes.
Retinoid receptors, transporters, and metabolizers as therapeutic targets in late onset Alzheimer disease
Ann B. Goodman
J. Cell. Physiol., Dec 2006; 209: 598–603. http://dx.doi.org:/10.1002/jcp.20784
Vitamin A (retinoid) is required in the adult brain to enable cognition, learning, and memory. While brain levels of retinoid diminish over the course of normal ageing, retinoid deficit is greater in late onset Alzheimer disease (LOAD) brains than in normal-aged controls. This paper reviews recent evidence supporting these statements and further suggests that genes necessary for the synthesis, transport and function of retinoid to and within the ageing brain are appropriate targets for treatment of LOAD. These genes tend to be clustered with genes that have been proposed as candidates in LOAD, are found at chromosomal regions linked to LOAD, and suggest the possibility of an overall coordinated regulation. This phenomenon is termed Chromeron and is analogous to the operon mechanism observed in prokaryotes. Suggested treatment targets are the retinoic-acid inactivating enzymes (CYP26)s, the retinol binding and transport proteins, retinol-binding protein (RBP)4 and transthyretin (TTR), and the retinoid receptors. TTR as a LOAD target is the subject of active investigation. The retinoid receptors and the retinoid-inactivating enzymes have previously been proposed as targets. This is the first report to suggest that RBP4 is an amenable treatment target in LOAD. RBP4 is elevated in type-2 diabetes and obesity, conditions associated with increased risk for LOAD. Fenretinide, a novel synthetic retinoic acid (RA) analog lowers RBP4 in glucose intolerant obese mice. The feasibility of using fenretinide either as an adjunct to present LOAD therapies, or on its own as an early prevention strategy should be determined.
Transthyretin: a key gene involved in the maintenance of memory capacities during aging.
Brouillette J1, Quirion R.
Neurobiol Aging. 2008 Nov; 29(11): 1721-32. Epub 2007 May 23.
Aging is often associated with decline of memory function. Aged animals, like humans, can naturally develop memory impairments and thus represent a useful model to investigate genes involved in long-term memory formation that are differentially expressed between aged memory-impaired (AI) and aged memory-unimpaired (AU) animals following stimulation in a spatial memory task. We found that alterations in hippocampal gene expression of transthyretin (TTR), calcineurin, and NAD(P)H dehydrogenase quinone 2 (NQO2) were associated with memory deficits in aged animals. Decreased TTR gene expression could be attributed at least partially to diminish activity of C/EBP immediate-early gene cascade initiated by CREB since protein levels of C/EBP, a transcription factor regulating both TTR and NQO2 expression, was decreased in AI animals. Memory deficits were also found during aging in mice lacking TTR. Treatment with retinoic acid reversed cognitive deficits in these knock-out mice as well as in aged rats. Our study provides genetic, behavioural and molecular evidence that TTR is involved in the maintenance of normal cognitive processes during aging by acting on the retinoid signaling pathway.
Neuroprotective effects of natural products: interaction with intracellular kinases, amyloid peptides and a possible role for transthyretin.
Bastianetto S1, Brouillette J, Quirion R.
Neurochem Res. 2007 Oct;32(10):1720-5. Epub 2007 Apr 4.
Various studies reported on the neuroprotective effects of natural products, particularly polyphenols, widely present in food and beverages. For example, we have shown that resveratrol, a polyphenol contained present in red wine and other foods, activates the phosphorylation of protein kinase C (PKC), this effect being involved in its neuroprotective action against Ass-induced toxicity. Moreover, tea-derived catechin gallate esters inhibit the formation Ass oligomers/fibrils, suggesting that this action likely contributes to their neuroprotective effects. Interestingly, the effects of polyphenols may be attributable, at least in part, to the presence of specific binding sites. Autoradiographic studies revealed that these binding sites are particularly enriched in choroids plexus in the rat brain. Interestingly, the choroid plexus secretes transthyretin, a protein that has been shown to prevent Abeta aggregation and that may be critical to the maintenance of normal learning capacities in aging. Taken together, these data suggest that polyphenols target multiple enzymes/proteins leading to their neuroprotective actions.
Aboard Transthyretin: From Transport to Cleavage
MA Liz, FM Mar, F Franquinho and MM Sousa
IUBMB Life 2010; 62(6): 429–435.
Transthyretin (TTR) is a plasma and cerebrospinal fluid protein mainly recognized as the transporter of thyroxine (T4) and retinol. Mutated TTR leads to familial amyloid polyneuropathy, a neurodegenerative disorder characterized by TTR amyloid deposition particularly in peripheral nerves. Beside its transport activities, TTR is a cryptic protease and participates in the biology of the nervous system. Several studies have been directed at finding new ligands of TTR to further explore the biology of the protein. From the identified ligands, some were in fact TTR protease substrates. In this review, we will discuss the existent information concerning TTR ligands/substrates.
The amyloidogenic potential of transthyretin variants correlates with their tendency to aggregate in solution
A Quintasa, MJM Saraiva, RMM Brito
FEBS Letters 1997; 418: 297-300
Amyloid fibril formation and deposition are the basis for a wide range of diseases, including spongiform encephalopathies, Alzheimer’s and familial amyloidotic polyneuropathies. However, the molecular mechanisms of amyloid formation are still poorly characterized. In certain forms of familial amyloidotic polyneuropathy (FAP), the amyloid fibrils are mostly constituted by variants of transthyretin (TTR). V30M-TTR is the most frequent variant, and L55P-TTR is the variant associated with the most aggressive form of amyloidosis. Here, we report gel filtration chromatography experiments to characterize the aggregation states of WT-, V30M-, L55P-TTR and a non-amyloidogenic variant, T119M-TTR, in solution, at nearly physiological pH. These studies show that all four protein tetramers dissociate to monomer upon dilution, in the submicromolar range, at pH 7.0. The amyloidogenic proteins V30M- and L55P-TTR show a complex equilibrium between monomers, tetramers and high molecular weight aggregate species. These aggregates dissociate directly to monomer upon dilution. This study shows that the tendency to form aggregates among the four studied proteins correlates with their known amyloidogenic potential. Thus, the amyloidogenic mutations could perturb the structure and/or stability of the monomeric species leading initially to the formation of soluble aggregates and at a later stage to insoluble amyloid fibrils.
Plasma Transthyretin Indicates the Direction of both Nitrogen Balance and Retinoid Status in Health and Disease
Ingenbleek Y and Bienvenu J
The Open Clinical Chemistry Journal, 2008; 1: 1-12
Whatever the nutritional status and the disease condition, the actual transthyretin (TTR) plasma level is determined by opposing influences between anabolic and catabolic alterations. Rising TTR values indicate that synthetic processes prevail over tissue breakdown with a nitrogen balance (NB) turning positive as a result of efficient nutritional support and / or anti-inflammatory therapy. Declining TTR values point to the failure to sustain NB as an effect of maladjusted dietetic management and / or further worsening of the morbid condition. Serial measurement of TTR thus appears as a dynamic index defining the direction of NB in acute and chronic disorders. The level of TTR production by the liver also works as a limiting factor for the cellular bioavailability of retinol and retinoid derivatives which play major roles in the brain ageing process. Optimal protein nutritional status, as assessed by TTR values within the normal range, prevents the occurrence of vascular and cerebral damages while maintaining the retinoid-mediated memory, cognitive and behavioral activities of elderly persons.
In Vitro and In Vivo Interactions of Homocysteine with Human Plasma Transthyretin
A Lim, S Sengupta, ME McComb, R Théberge, WG Wilson, CE Costello, DW Jacobsen
J. Biol. Chem. Sep 24, 2003. Manuscript M306748200 http://dx.doi.org:/10.1074/jbc.M306748200
Hyperhomocysteinemia is an independent risk factor for cardiovascular disease and an emerging risk factor for cognitive dysfunction and Alzheimer’s disease. Greater than 70% of the homocysteine in plasma is disulfide bonded to protein cysteine residues. The identity and functional consequences of protein homocysteinylation are just now emerging. The amyloidogenic protein transthyretin (prealbumin), as we now report, undergoes homocysteinylation at its single cysteine residue (Cys10) both in vitro and in vivo. Thus, when human plasma or highly purified transthyretin was incubated with 35SLhomocysteine followed by SDSPAGE and phosphor-imaging, two bands corresponding to transthyretin dimer and tetramer were observed. Treatment of the labeled samples with βmercaptoethanol prior to SDSPAGE removed the disulfide bound homocysteine. Transthyretin-Cys10SS-homocysteine
was then identified in vivo in plasma from normal donors, patients with end stage renal disease and homocystinurics by immune-precipitation and HPLC/electrospray mass spectrometry. The ratios of transthyretin-Cys10SS-homocysteine and transthyretin-Cys10SS-sulfonate to that of unmodified transthyretin increased with increasing homocysteine plasma concentrations whereas the ratio of transthyretin-Cys10SS-cysteine to that of unmodified transthyretin decreased. Hyperhomocystein-emic burden is thus reflected in the plasma levels of transthyretin-Cys10SS-homocysteine, which in turn may contribute to the pathological consequences of amyloid disease.
Support for the multigenic hypothesis of amyloidosis: The binding stoichiometry of retinol-binding protein, vitamin A, and thyroid hormone influences transthyretin amyloidogenicity in vitro
Joleen T. White and Jeffery W. Kelly
PNAS Nov 6, 2001; 98(23): 13019–13024 http://pnas/cgi/dx.doi.org:/10.1073/pnas.241406698
The amyloidoses are a large group of protein misfolding diseases.
Genetic and biochemical evidence support the hypothesis that amyloid formation from wild-type or 1 of 80 sequence variants of transthyretin causes the human amyloid diseases senile systemic amyloidosis or familial amyloid polyneuropathy, respectively. The late onset and variable penetrance of these diseases has led to their designation as multigenic—implying that the expression levels and alleles of multiple gene products influence the course of pathology. Here we show that the binding stoichiometry of three interacting molecules, retinol-binding protein, vitamin A, and L-thyroxine, notably influenced transthyretin amyloidogenicity in vitro. At least 70 genes control retinol-binding protein, vitamin A, and L-thyroxine levels in plasma and have the potential to modulate the course of senile systemic amyloidosis or familial amyloid polyneuropathy.
Transthyretin sequesters amyloid protein and prevents amyloid formation
AL Schwarzman, L Gregori, MP Vitek, S Lyubski, et al.
Proc. Natl. Acad. Sci. USA Aug 1994; 91: 8368-8372. Neurobiology
The cardinal pathological features of Alzheimer disease are depositions of aggregated amyloid 13 protein (AP) in the brain and cerebral vasculature. However, the AP3 is found in a soluble form in cerebrospinal fluid in healthy individuals and patients with Alzheimer disease. We postulate that sequestration of AP3 precludes amyloid formation. Failure to sequester AP in Alzheimer disease may result in amyloidosis. When we added AP3 to cerebrospinal fluid of patients and controls it was rapidly sequestered into stable complexes with transthyretin. Complexes with apolipoprotein E, which has been shown to bind AP3 in vitro, were not observed in cerebrospinal fluid. Additional in vitro studies showed that both purified transthyretin and apolipoprotein E prevent amyloid formation.
Oxytocin
Variation in the oxytocin receptor gene is associated with behavioral and neural correlates of empathic accuracy
HR Laursen, HR Siebner, T Haren, K Madsen, R Grønlund, O Hulme, S Henningsson
Front Behavioral Neurosci 6 Dec 2014; 8(423): 1-10.
http://dx.doi.org:/10.3389/fnbeh.2014.00423
The neuromodulators oxytocin and serotonin have been implicated in regulating affective processes underlying empathy. Understanding this dependency, however, has been limited by a lack of objective metrics for measuring empathic performance. Here we employ a novel psychophysical method for measuring empathic performance that quantitatively measures the ability of subjects to decode the experience of another person’s pain. In 50 female subjects, we acquired functional magnetic resonance imaging data as they were exposed to a target subject experiencing variable degrees of pain, whilst performing an irrelevant attention-demanding task. We investigated the effect of variation in the oxytocin receptor gene (OXTR) and the serotonin transporter gene (SLC6A4) on the psychophysical and neurometric variability associated with empathic performance. The OXTR rs2268498 and rs53576 polymorphisms, but not the SLC6A45-HTTLPR, were associated with significant differences in empathic accuracy, with CC-and AA-carriers, respectively, displaying higher empathic accuracy. For OXTR rs2268498 there was also a genotype difference in the correlation between empathic accuracy and activity in the superior temporal sulcus (STS). In OXTR rs2268498CC-carriers, high empathic accuracy was associated with stronger responsiveness of the right STS to the observed pain. Together, the results show that genetic variation in the OXTR has significant influence on empathic accuracy and that this may be linked to variable responsivity of the STS.
Naturally occurring variations in maternal behavior in the rat are associated with differences in estrogen inducible central oxytocin receptors
Frances Champagne, Josie Diorio, Shakti Sharma, and Michael J. Meaney
PNAS Oct 23, 2001; 98(22): 12736–12741.
http://pnas.org/cgi/doi/10.1073/pnas.221224598
Naturally occurring variations in maternal licking/grooming influence neural development and are transmitted from mother to female offspring. We found that the induction of maternal behavior in virgin females through constant exposure to pups (pup sensitization) was significantly shorter in the offspring of High compared with Low licking/grooming mothers, suggesting differences in maternal responsivity. In randomly selected females screened for individual differences in maternal responsivity and subsequently mated, there was a significant and negative correlation (r520.73) between the latency to exhibit maternal behavior in the pup sensitization paradigm and the frequency of pup licking/grooming during lactation. Females that were more maternally responsive to pups and that showed increased levels of pup licking/grooming also showed significantly higher oxytocin receptor levels in the medial preoptic area, the lateral septum, the central nucleus (n.) of the amygdala, the paraventricular n. of the hypothalamus, and the bed n. of the stria terminalis. Intra-cerebroventricular administration of an oxytocin receptor antagonist to mothers on postpartum day 3 completely eliminated the differences in pup licking/grooming, suggesting that differences in oxytocin receptor levels are functionally related to maternal behavior. Finally, estrogen treatment of virgin females significantly increased oxytocin receptor binding in the medial preoptic area and lateral septum of female offspring of High, but not Low, licking/grooming mothers. These findings suggest that maternal licking/grooming influences the development of estrogen sensitivity in brain regions that regulate maternal behavior, providing a potential mechanism for the intergenerational transmission of individual differences in maternal behavior.
Life in groups: the roles of oxytocin in mammalian sociality
Allison M. J. Anacker and Annaliese K. Beery
Front Behavioral Neurosci Dec2013; 7(185): 1 – 10.
http://dx.doi.org:/10.3389/fnbeh.2013.00185
In recent decades, scientific understanding of the many roles of oxytocin (OT) in
social behavior has advanced tremendously. The focus of this research has been on maternal attachments and reproductive pair-bonds, and much less is known about the substrates of sociality outside of reproductive contexts. It is now apparent that OT influences many aspects of social behavior including recognition, trust, empathy, and other components of the behavioral repertoire of social lspecies. This review provides a comparative perspective on the contributions of OT to life in mammalian social groups. We provide background on the functions of OT in maternal attachments and the early social lenvironment, and give an overview of the role of OT circuitry in support of different mating systems. We then introduce peer relationships in group-living rodents as a means for studying the importance of OT in non-reproductive affiliative behaviors. We review species differences in oxytocin receptor (OTR )distributions in solitary and group-living species of South American tuco-tucos and in African mole-rats, as well as singing mice. We discuss variation in OTR levels with seasonal changes in social behavior in female meadow voles, and the effects of OT manipulations on peer huddling behavior. Finally, we discuss avenues of promise for future investigation, and relate current findings to research in humans and non-human primates. There is growing evidence that OT is involved in social selectivity, including increases in aggression toward social outgroups and decreased huddling with unfamiliar individuals, which may support existing social structures or relationships at the expense of others. OT’s effects reach beyond maternal attachment and pair bonds to play a role in affiliative behavior underlying “friendships”, organization of broad social structures, and maintenance of established social relationships with individuals or groups.
Relative conformational rigidity in oxytocin and [1-penicillamine]-oxytocin: A proposal for the relationship of conformational flexibility to peptide hormone agonism and antagonism
Jean-Paul Meraldi, Victor J. Hruby, and Anne I. Richard Brewster
Proc. Natl. Acad. Sci. USA Apr 1977; 74(4): 1373-1377, Biochemistry
A comparative study of the proton and carbon-13 nuclear magnetic resonance spectral parameters of the peptide hormone oxytocin and of its competitive inhibitor [1-L-penicillamineloxytocin has been made, and the results analyzed in terms of comparative conformational and dynamic properties. The results indicate that oxytocin has a flexible conformation, while [1-L-penicillamineJoxytocin has a more restricted conformation. The results provide a framework for understanding the mechanism of peptide hormone agonism and antagonism for these compounds, and an approach for understanding some features of the interaction of the hormone and related compounds with their receptor.
Relation of the Conformation of Oxytocin to the Biology of Neurohypophyseal Hormones
Roderich Walter, I. L. Schwartz, J. H. Darnell, And D. W. Urry
Proc. Nat. Acad. Sci. USA Jun 1971; 68(6): 1355-1359.
The conformation of oxytocin is related to the evolution and to some of the biological activities of neurohypophyseal hormonal peptides. On the basis of the three-dimensional structure, positions 3, 4, 7, and 8 are the only loci at which naturally occurring neurohypophyseal peptides may be expected to differ. The side chains of these amino-acid residues are the primary determinants of the differential specificity in interactions between neurohypophyseal hormones and their receptors.
There are three general groups of structural modifications of neurohypophyseal hormones which can be correlated with specific changes in biological activity: (a) those affecting the stabilization of the backbone of the peptide, which would extensively perturb the spatial relationships among all the constituent amino acids and hence, affect both affinity and intrinsic activities uniformly; (b) those which, while retaining the stability of the backbone conformation, alter the steric environment and charge distribution of limited surface areas, and thereby can affect affinity and intrinsic activity differentially; and (c) those changing the steric and electronic requirements of moieties comprising the active surface of the neurohypophyseal peptide, without perturbing the peptide backbone of the hormone molecule and, hence, affecting intrinsic activity without altering affinity.
The Nonpeptide Oxytocin Receptor Agonist WAY 267,464: Receptor-Binding Profile, Prosocial Effects and Distribution of c-Fos Expression in Adolescent Rats
C. Hicks, W. Jorgensen, C. Brown, J. Fardell, J. Koehbach, C. W. Gruber, et al.
J Neuroendocrinol. Jul 2012; 24(7): 1012–1029.
http://dx.doi.org:/10.111/j.1365-2826.2012.02311.x.
Previous research suggests that the nonpeptide oxytocin receptor (OTR) agonist WAY 267,464 may only partly mimic the effects of oxytocin in rodents. The present study further explored these differences and related them to OTR and vasopressin 1a receptor (V1aR) pharmacology and regional patterns of c-Fos expression. Binding data for WAY 267,464 and oxytocin were obtained by displacement binding assays on cellular membranes, while functional receptor data were generated by luciferase reporter assays. For behavioral testing, adolescent rats were tested in a social preference paradigm, the elevated plus-maze (EPM) and for locomotor activity changes following WAY 267,464 (10 and 100 mg/kg, i.p.) or oxytocin (0.1 and 1 mg/kg, i.p.). The higher doses were also examined for their effects on regional c-Fos expression. Results showed that WAY 267,464 had higher affinity (Ki) at the V1aR than the OTR (113 versus 978 nM). However, it had no functional response at the V1aR and only a weak functional effect (EC50) at the OTR (881 nM). This suggests WAY 267,464 is an OTR agonist with weak affinity and a possible V1aR antagonist. Oxytocin showed high binding at the OTR (1.0 nM) and V1aR (503 nM), with a functional EC50 of 9.0 and 59.7 nM, respectively, indicating it is a potent OTR agonist and full V1aR agonist. WAY 267,464 (100 mg/kg), but not oxytocin, significantly increased the proportion of time spent with a live rat, over a dummy rat, in the social preference test. Neither compound affected EPM behaviour, whereas the higher doses of WAY 267,464 and oxytocin suppressed locomotor activity. WAY 267,464 and oxytocin produced similar c-Fos expression in the paraventricular hypothalamic nucleus, central amygdala, lateral parabrachial nucleus and nucleus of the solitary tract, suggesting a commonality of action at the OTR with the differential doses employed. However, WAY 267,464 caused greater c-Fos expression in the medial amygdala and the supraoptic nucleus than oxytocin, and lesser effects in the locus coeruleus. Overall, our results confirm the differential effects of WAY 267,464 and oxytocin and suggest that this may reflect contrasting actions of WAY 267,464 and oxytocin at the V1aR. Antagonism of the V1aR by WAY.
Direct identification of human oxytocin receptor- binding domains using a photoactivatable cyclic peptide antagonist: Comparison with the human V1a vasopressin receptor
C Breton, H Chelli, M Kabbaj-Benmansour, E Carnazzi, et al.
J. Biol. Chem. May 3, 2001 M102073200
http://dx.doi.org:/10.1074/jbc.M102073200
Understanding of the molecular determinants responsible for antagonist binding to the oxytocin receptor should provide important insights that facilitate rational design of potential therapeutic agents for the treatment of preterm labor. To study ligand-receptor interactions, we used a novel photosensitive radio-iodinated antagonist of the human oxytocin receptor, termed [125I]-ZOTA. This ligand had an equivalent high affinity for human oxytocin and V1a vasopressin receptors expressed in CHO cells. Taking advantage of this dual specificity, we conducted photo-affinity labeling experiments on both receptors. Photo-labeled oxytocin and V1a receptors appeared as a unique protein band at 70-75 kDa and two labeled protein bands at 85-90 kDa and 46 kDa, respectively. To identify contact sites between the antagonist and the receptors, the labeled 70-75 kDa and the 46 kDa proteins were cleaved with CNBr and digested with Lys-C and Arg-C endoproteinases. The fragmentation patterns allowed to identify a covalently labeled region in the oxytocin receptor transmembrane domain III, consisting of the residues Leu114-Val115-Lys116. Analysis of contact sites in the V1a receptor led to the identification of the homologous region, consisting of the residues Val126-Val127-Lys128. Binding domains were confirmed by mutation of several CNBr cleavage sites in the oxytocin receptor and of one Lys-C cleavage site in the V1a receptor. The results are in agreement with previous experimental data and three-dimensional models of agonist and antagonist binding to members of the oxytocin/vasopressin receptor family.
Presynaptic modulation by metabotropic glutamate receptors of excitatory and inhibitory synaptic inputs to hypothalamic magnocellular neurons.
Schrader, L. A. and J. G. Tasker.
J. Neurophysiol. 1997; 77: 527–536.
The effects of activation of metabotropic glutamate receptors (mGluRs) on synaptic inputs to magnocellular neurons of the hypothalamic supraoptic nucleus (SON) were studied with the use of whole cell patch-clamp and microelectrode recordings in acute hypothalamic slices.
These data indicate that mGluRs in the hypothalamus have opposing actions at somata/dendrites and at presynaptic terminals. Activa- tion of group I receptors (mGluR1 and/or mGluR5) on presynaptic somata/dendrites led to an increase in spike-dependent transmitter release, whereas activation of the group III receptors (mGluR4, 7, and/or 8) on presynaptic terminals suppressed glutamate and GABA release onto SON neurons. No diffferences were seen in the effects of mGluR activation between immunohistochemically oxytocin and vasopressin neurons of the SON.
Postsynaptic mechanism of depression of GABAergic synapses by oxytocin in the supraoptic nucleus of immature rat
A. B. Brussaard, K. S. Kits and T. A. de Vlieger
Journal of Physiology 1996; 497(2): 495-507
- Oxytocin is known to act on autoreceptors of oxytocin neurons in the supraoptic nucleus (SON). We investigated whether oxytocin modulates putative oxytocin neurones by suppressing the GABAA receptor-mediated synaptic inputs on these cells.
- GABAergic inhibitory postsynaptic currents (IPSCs) were recorded from SON neurons in hypothalamic slices from young rats. Oxytocin specifically reduced the amplitude of both spontaneous and evoked IPSCs, without altering their current kinetics.
- The effect of oxytocin was observed in 70% of the magnocellular neurons recorded from the dorsomedial part of the SON. d(CH2)5OVT, a specific antagonist of oxytocin receptors, blocked the effect of oxytocin on the IPSCs. Vasopressin had no effect on oxytocin-sensitive SON neurons.
- The intervals between spontaneous IPSCs were not affected by oxytocin. This suggested that oxytocin had a postsynaptic effect on SON neurons.
- This postsynaptic origin was further substantiated by application of TTX, which blocked all evoked release but did not prevent the suppressive effect of oxytocin on the amplitude of the spontaneous IPSCs still present in the recording. The selective effect of oxytocin on IPSC amplitude was also maintained in nominally zero extracellular calcium.
- Intracellulax perfusion of SON neurones with GTPyS mimicked the effect of oxytocin on IPSCs, while GDP/6S, similarly applied, abolished the effect of oxytocin.
- Application of calcium mobilizers such as thapsigargin and caffeine also reduced the amplitude of spontaneous IPSCs without significantly altering the frequency at which IPSCs occurred.
- Thus, oxytocin depresses GABAergic synapses in the SON via modulation of the postsynaptic GABAA receptors. This would lead to disinhibition of SON neurons sensitive to oxytocin and could, therefore, be a powerful means of controlling the firing of oxytocin neurons.
Oxytocin, vasopressin and sociality
CS Carter, AJ Grippo, H Pournajafi-Nazarloo, MG Ruscio and SW Porges
Progress in Brain Research 2008; 170:331-36.
http://dx. doi.org:/10.1016/S0079-6123(08)00427-5
The neurobiology of social behavior is interwoven with autonomic, endocrine and other homoeostatic processes responsible for the adaptive functions of reproduction and survival. Young mammals are dependent on their mothers for nourishment, and the interaction between the mother and infant may be a physiological and neuroendocrine prototype for mammalian sociality. Although these adaptive functions of the mother–infant social behavioral dyad are obvious, adult social interactions, including social bonds, also are important to health and survival. Two neuropeptides, oxytocin (OXT) and arginine vasopressin (AVP), have been repeatedly implicated in mammalian social behaviours and emotional states that support sociality. Although best known for their roles in reproduction and homoeostasis, these peptides play a central role in the activation and expression of social behaviours and emotional states. Recent studies from our work with the prairie vole (Microtus ochrogaster), reviewed here, reveal a role for both OXT and AVP in behavioral and endocrine changes during social interactions, and also changes that are associated with the absence of social interactions (i.e. social isolation).
Serotonergic Neurotransmission in Autism Spectrum Disorders, Autism – A Neurodevelopmental Journey from Genes to Behaviour, Dr. Valsamma Eapen (Ed.), ISBN: 978-953-307-493-1, InTech, Available from: http://www.intechopen.com/books/autism-a-neurodevelopmental-journey-fromgenes-to-behaviour/serotonergic-neurotransmission-in-autism-spectrum-disorders
Chapter 5. Serotonergic Neurotransmission in Autism Spectrum Disorders
Yoshihiro Takeuchi
3.7 Oxytocin and autism spectrum disorders, pp96-97
Oxytocin is a peptide hormone produced by neurosecretory cells at the hypothalamus supraoptic nucleus and paraventricular nucleus in mothers and it is secreted from the posterior pituitary lobe. The hormone exhibits a variety of functions in the brain, as well as roles in delivery and galactopoiesis. Oxytocin regulates emotion in the company of somebody and oxytocin concentrations in the blood are low in autism spectrum disorders patients, therefore, the hormone attracts the most attention in investigations of the pathogenesis of autism spectrum disorders (Kirsch. et al; 2005). Oxytocin plays a role in signal transmission between the mother and the fetus so that neurons in the fetus are prepared for delivery. Oxytocin temporarily switches intracerebral GABAergic neurotransmission from the excitatory to inhibitory state in the fetus at delivery and exerts neuroprotective action. Serotonin fiber endings are abundant on oxytocin neurons at the hypothalamus supraoptic nucleus and paraventricular nucleus, and it should be noted that oxytocin neurons are regulated by the serotonin neuron system.
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