POU-domain gene expression in the gastrointestinal tract

The process of self-renewal which occurs in the gastrointestinal epithelium is greatly amplified and accelerated during the intestinal adaptation which occurs in the residual ileum after massive small bowel resection (MSBR). As with growth and development, these processes must involve the coordinated regulation of many genes. Several families of nuclear proteins are known to be involved in the control of gene expression during development including the POU-domain genes; their expression has not been characterized in the gastrointestinal tract during normal cellular renewal or adaptation, and POU-domain encoding cDNAs were cloned from ileal RNA. Three known genes were cloned: Oct-1, Brn-1 and Tst-1 but no novel members of this gene family were identified. The encoded sequence for rat Oct-1 differs from that previously reported. Oct-1 is relatively ubiquitously expressed with increased expression during both development and adaptation. Minimal expression of Tst-1 was observed. Brn-1 exhibits limited expression in the adult gastrointestinal tract. but may play a role in the fetal gastrointestinal tract.     

Analysis of expression and posttranslational modification of proteins during hypoxia.

The cellular responses to hypoxia are complex and characterized by alterations in the expression of a number of genes, including stress-related genes and corresponding proteins that are necessary to maintain homeostasis. The purpose of this article is to review previous and recent studies that have examined the changes in the expression and posttranslational modification of proteins in response to chronic sustained and intermittent forms of hypoxia. A large number of studies focused on the analysis of either the single protein or a subset of related proteins using one-dimensional gel electrophoresis to separate a complex set of proteins from solubilized tissues or cell extracts, followed by immunostaining of proteins using antibodies that are specific to either native or posttranslationally modified forms. On the other hand, only a limited number of studies have examined the global perturbations on protein expression by hypoxia using proteomics approach involving two-dimensional electrophoresis coupled with mass spectrometry. Results derived from specific protein analysis of a variety of tissues and cells showed that hypoxia, depending on the duration and severity of the stimulus, affects the level and the state of posttranslational modification of a subset of proteins that are associated with energy metabolism, stress response, cell injury, development, and apoptosis. Some of these earlier findings are further corroborated by recent studies that utilize a global proteomics approach, and, more importantly, results from these proteomics investigations on the effects of hypoxia provide new protein targets for further functional analysis. The anticipated new information stems from the analysis of expression, and posttranslational modification of these novel protein targets, along with gene expression profiles, offers exciting new opportunities to further define the mechanisms of cellular responses to hypoxia and to control more effectively the clinical consequences of prolonged or periodic lack of oxygen.     

Kallikrein-gene expression in the rat gastrointestinal tract.

The serine proteinase glandular kallikrein has been demonstrated in the gastrointestinal tract, although there is some doubt as to whether it is synthesized there or derives from exocrine-gland secretions. Using a rat pancreatic kallikrein cRNA probe we have demonstrated kallikrein-like gene expression in the corpus, duodenum, jejunum, ileum, caecum and colon, and compared the pattern of expression with that of the gastrointestinal peptides somatostatin, gastrin and glucagon. In addition, using a panel of oligonucleotide probes specific for various members of the rat kallikrein-gene family, we have shown that the kallikrein-like gene expressed appears to be expressed as true kallikrein.     

The expression and posttranslational modification of a neuron-specific beta-tubulin isotype during chick embryogenesis

Five beta-tubulin isotypes are expressed differentially during chicken brain development. One of these isotypes is encoded by the gene c beta 4 and has been assigned to an isotypic family designated as Class III (beta III). In the nervous system of higher vertebrates, beta III is synthesized exclusively by neurons. A beta III-specific monoclonal antibody was used to determine when during chick embryogenesis c beta 4 is expressed, the cellular localization of beta III, and the number of charge variants (isoforms) into which beta III can be resolved by isoelectric focusing. On Western blots, beta III is first detectable at stages 12-13. Thereafter, the relative abundance of beta III in brain increases steadily, apparently in conjunction with the rate of neural differentiation. The isotype was not detectable in non-neural tissue extracts from older embryos (days 10-14) and hatchlings. Western blots of protein separated by two-dimensional gel electrophoresis (2D-PAGE) reveal that the number of beta III isoforms increases from one to three during neural development. This evidence indicates that beta III is a substrate for developmentally regulated, multiple-site posttranslational modification. Immunocytochemical studies reveal that while c beta 4 expression is restricted predominantly to the nervous system, it is transiently expressed in some embryonic structures. More importantly, in the nervous system, immunoreactive cells were located primarily in the non-proliferative marginal zone of the neural epithelia. Regions containing primarily mitotic neuroblasts were virtually unstained. This localization pattern indicates that c beta 4 expression occurs either during or immediately following terminal mitosis, and suggests that beta III may have a unique role during early neuronal differentiation and neurite outgrowth.     

Distribution of glucagon-like peptide I in canine and feline pancreas and gastrointestinal tract

Recently, a putative hormone, glucagon-like peptide I (GLP I), has been identified in the predicted sequences of the precursors to pancreatic glucagon in human, rat, hamster, and ox. The distribution of GLP I immunoreactivity in canine and feline pancreas and gastrointestinal tract was examined immunohistochemically and was compared with that of two other antigenic determinants of pancreatic pro-glucagon, i.e., glucagon and the NH2 terminus of glicentin. All three determinants occurred in the same population of islet cells in normal pancreas and in pancreas consisting predominantly of islet tissue from dogs with canine pancreatic acinar atrophy. Northern blot analysis of mRNA from the latter tissue, using a rat pre-pro-glucagon complementary DNA probe, revealed a single mRNA species similar in size to the pre-pro-glucagon mRNA detected in fetal rat pancreas. The three antigenic determinants of pancreatic pro-glucagon were co-localized also in intestinal L-cells and in canine gastric A-cells. Canine and feline pancreatic pro-glucagons therefore resemble those identified in other mammals and may also occur in gastrointestinal endocrine cells. Although there is evidence that the GLP I sequence is not liberated from pancreatic pro-glucagon, our results raise the possibility that this putative hormone may be a cleavage product of pro-glucagon in the gastrointestinal tract.     

Rho Family GTPase modification and dependence on CAAX motif-signaled posttranslational modification

Rho GTPases (20 human members) comprise a major branch of the Ras superfamily of small GTPases, and aberrant Rho GTPase function has been implicated in oncogenesis and other human diseases. Although many of our current concepts of Rho GTPases are based on the three classical members (RhoA, Rac1, and Cdc42), recent studies have revealed the diversity of biological functions mediated by other family members. A key basis for the functional diversity of Rho GTPases is their association with distinct subcellular compartments, which is dictated in part by three posttranslational modifications signaled by their carboxyl-terminal CAAX (where C represents cysteine, A is an aliphatic amino acid, and X is a terminal amino acid) tetrapeptide motifs. CAAX motifs are substrates for the prenyltransferase-catalyzed addition of either farnesyl or geranylgeranyl isoprenoid lipids, Rce1-catalyzed endoproteolytic cleavage of the AAX amino acids, and Icmt-catalyzed carboxyl methylation of the isoprenylcysteine. We utilized pharmacologic, biochemical, and genetic approaches to determine the sequence requirements and roles of CAAX signal modifications in dictating the subcellular locations and functions of the Rho GTPase family. Although the classical Rho GTPases are modified by geranylgeranylation, we found that a majority of the other Rho GTPases are substrates for farnesyltransferase. We found that the membrane association and/or function of Rho GTPases are differentially dependent on Rce1- and Icmt-mediated modifications. Our results further delineate the sequence requirements for prenyltransferase specificity and functional roles for protein prenylation in Rho GTPase function. We conclude that a majority of Rho GTPases are targets for pharmacologic inhibitors of farnesyltransferase, Rce1, and Icmt.     

Citrullination: a posttranslational modification in health and disease

Posttranslational modifications are chemical changes to proteins that take place after synthesis. One such modification, peptidylarginine to peptidylcitrulline conversion, catalysed by peptidylarginine deiminases, has recently received significant interest in biomedicine. Introduction of citrulline dramatically changes the structure and function of proteins. It has been implicated in several physiological and pathological processes. Physiological processes include epithelial terminal differentiation, gene expression regulation, and apoptosis. Rheumatoid arthritis, multiple sclerosis, and Alzheimer's disease are examples of human diseases where protein citrullination involvement has been demonstrated. In this review, we discuss our current understanding on the importance of protein deimination in these processes. We describe the enzymes catalyzing the reaction, as well as their known protein substrates. We review the citrullinated peptide epitopes that are proposed as disease markers, specifically recognized in certain human autoimmune disorders. The potential autopathogenic role of citrullinated epitopes is also discussed.     

Mineralocorticoid receptor gene expression in the gastrointestinal tract: distribution and ontogeny

The gastrointestinal tract is a well characterized target tissue for aldosterone, where it regulates electrolyte transport, particularly in the descending colon. Previous studies have demonstrated the presence of aldosterone receptors in gastrointestinal tissues. We have used specific cRNA probes for the rat mineralocorticoid receptor to explore both the distribution and ontogeny of mineralocorticoid receptor gene expression in the gastrointestinal tract. Mineralocorticoid receptor gene expression is found throughout the small and large intestine, but is absent from the stomach. The highest levels are observed in the distal colon, and significant expression is found in the duodenum; in both tissues levels of expression are higher than those in kidney. In both the developing duodenum and colon, mineralocorticoid receptor gene expression precedes the development of the full physiological response to aldosterone. These findings emphasise the colon as an important target tissue for aldosterone, and raise the question of potential roles for aldosterone in the duodenum.     

The posttranslational modification of thyrotropin receptor and thyroid diseases

The thyrotropin receptor (TSHR), lutropin receptor, and follitropin receptor are related members of the superfamily of leucine-rich repeats containing adenylate cyclase stimulating receptors. The unique posttranslational modification of the TSHR leads to the transformation of its monomeric form to the subunit structure where the subunits A and B are connected by disulphide bonds. This natural processing occurs with the release from the receptor of a short peptide C, and is followed by the release of the subunit A. Both monomeric and dimeric forms of the receptor are stimulated by TSH, so no clear functional significance of TSHR modifications have been found. We can speculated that the processing of TSHR with the release of its large fragments contributes to the development of autoimmune diseases and production anti-TSH receptor autoantibodies. The extrathyroidal manifestations of Graves disease may also be related to metastasis of the autoimmune reaction to extrathyroidal sites via the released A subunit. The TSHR processing may, to some extent, be connected to the hyperthyroidism since the release of the subunit A from the receptor augmented the adenylate cyclase activity in the absence of TSH. According to the recent model of receptors action the TSHR is in equilibrium between the inactive (closed) and active (opened) conformations. In opened conformation it can associate with Gs protein and trigger the intracellular signal. TSH and stimulating autoantibodies preferentially bind to opened receptors and stabilizes them.     

Sensation in the gastrointestinal tract

There are similarities between sensation in the gastrointestinal tract (GI tract) and somatic sensation. This review concentrates on parasympathetic (vagal) components of GI sensation rather than the sympathetic (splanchnic) elements. A wide range of enteroceptors have been described over the whole length of the gut which subserve several different sensory modalities. Fibres from these enteroceptors project to the medulla, primarily to the nucleus of the solitary tract. In the medulla there is considerable integration of afferent information from different parts of the GI tract. Regulatory peptides are present both in the brain and in the GI tract. It is likely that these peptides may play a role in the modulation of sensory information in the medulla. Parallels may be drawn at a receptor level between somatic sensation and sensation in the GI tract. More centrally, sensory mechanisms relating to the gut seem less highly organized than in somatic sensation. This reduced influence of the central nervous system in GI tract sensation may be explained by the presence in the gut of a highly sophisticated intrinsic nervous system, the enteric nervous system, which pre-programmes many of the functions of the GI tract.     

Probing the transglutaminase-mediated, posttranslational modification of proteins during development

Sphaerechinus granularis eggs were fertilized in seawater in the presence of 0.2 mM dansylcadaverine, and development was allowed to take place with this compound in the medium. gamma-Glutamyldansylcadaverine, indicative of the utilization of the amine tracer by intrinsic transglutaminase, was isolated from the embryonic proteins, and identity of the product with the chemically synthesized gamma-glutamyl derivative of dansylcadaverine was confirmed. Covalent labeling of proteins occurring during development was examined by means of electrophoresis in NaDodSO4, followed by immunoblotting with an antibody that specifically recognized the dansyl hapten. There was an increase in the total uptake of the tracer at an essentially constant rate with each cell division, from 2- to 8- and 64-cell stages. Moreover, multiple protein labeling was evident in all specimens. The described concept of studying posttranslational modifications in vivo by transglutaminase through detection of the haptenic or specific ligand recognizable group of an incorporated small amine substrate will undoubtedly be of general utility for probing the functions of this family of enzymes in other cell types as well.     

Taste receptors in the gastrointestinal tract. V. Acid sensing in the gastrointestinal tract

Luminal acidity is a physiological challenge in the foregut, and acidosis can occur throughout the gastrointestinal tract as a result of inflammation or ischemia. These conditions are surveyed by an elaborate network of acid-governed mechanisms to maintain homeostasis. Deviations from physiological values of extracellular pH are monitored by multiple acid sensors expressed by epithelial cells and sensory neurons. Acid-sensing ion channels are activated by moderate acidification, whereas transient receptor potential ion channels of the vanilloid subtype are gated by severe acidosis. Some ionotropic purinoceptor ion channels and two-pore domain background K(+) channels are also sensitive to alterations of extracellular pH.     

Regulation of the water channel aquaporin-2 by posttranslational modification

The cellular functions of many eukaryotic membrane proteins, including the vasopressin-regulated water channel aquaporin-2 (AQP2), are regulated by posttranslational modifications. In this article, we discuss the experimental discoveries that have advanced our understanding of how posttranslational modifications affect AQP2 function, especially as they relate to the role of AQP2 in the kidney. We review the most recent data demonstrating that glycosylation and, in particular, phosphorylation and ubiquitination are mechanisms that regulate AQP2 activity, subcellular sorting and distribution, degradation, and protein interactions. From a clinical perspective, posttranslational modification resulting in protein misrouting or degradation may explain certain forms of nephrogenic diabetes insipidus. In addition to providing major insight into the function and dynamics of renal AQP2 regulation, the analysis of AQP2 posttranslational modification may provide general clues as to the role of posttranslational modification for regulation of other membrane proteins.