scholarly journals Amino Acids 563–566 of the Na+/H+ Exchanger Isoform 1 C-Terminal Cytosolic Tail Prevent Protein Degradation and Stabilize Protein Expression and Activity

2020 ◽  
Vol 21 (5) ◽  
pp. 1737 ◽  
Author(s):  
Xiuju Li ◽  
Debajyoti Dutta ◽  
Martin Jung ◽  
Richard Zimmermann ◽  
Larry Fliegel

Isoform one of the mammalian Na+/H+ exchanger is a plasma membrane protein that is ubiquitously present in humans. It regulates intracellular pH through the removal of one intracellular proton in exchange for a single extracellular sodium. It consists of a 500 amino acid membrane domain plus a 315 amino acid, C-terminal tail. We examined amino acids of the C-terminal tail that are important in the targeting and activity of the protein. A previous study demonstrated that stop codon polymorphisms can result in decreased activity, expression, targeting and enhanced protein degradation. Here, we determine elements that are critical in these anomalies. A series of progressive deletions of the C-terminal tail demonstrated a progressive decrease in activity and targeting, though these remained until a final drop off with the deletion of amino acids 563–566. The deletion of the 562LIAGERS568 sequence or the alteration to the 562LAAAARS568 sequence caused the decreased protein expression, aberrant targeting, reduced activity and enhanced degradation of the Na+/H+ exchanger (NHE1) protein. The 562LIAGERS568 sequence bound to other regions of the C-terminal cytosolic domain. We suggest this region is necessary for the activity, targeting, stability, and expression of the NHE1 protein. The results define a new sequence that is important in maintenance of NHE1 protein levels and activity.

2019 ◽  
Author(s):  
Marc Weber ◽  
Raul Burgos ◽  
Eva Yus ◽  
Jae-Seong Yang ◽  
Maria Lluch-Senar ◽  
...  

AbstractThe C-terminal sequence of a protein is involved in processes such as efficiency of translation termination and protein degradation. However, the general relationship between features of this C-terminal sequence and levels of protein expression remains unknown. Here, we identified C-terminal amino acid biases that are ubiquitous across the bacterial taxonomy (1582 genomes). We showed that the frequency is higher for positively charged amino acids (lysine, arginine) while hydrophobic amino acids and threonine are lower. In highly abundant proteins, the C-terminal residue is more conserved. We then studied the impact of C-terminal composition on protein levels in a library of M. pneumoniae mutants, covering all possible combinations of the two last codons. We found that charged and polar residues, in particular lysine, led to higher expression, while hydrophobic and aromatic residues led to lower expression, with a difference in protein levels up to 4-fold. Our results demonstrate that the identity of the last amino acids has a strong influence on protein expression levels and is under selective pressure in highly expressed proteins.


2021 ◽  
Vol 22 (21) ◽  
pp. 11349
Author(s):  
Xiuju Li ◽  
Tommy Tu ◽  
Sicheng Quan ◽  
Francisco J. Quintero ◽  
Richard Fahlman ◽  
...  

The mammalian Na+/H+ exchanger isoform 1 (NHE1) is a plasma membrane protein ubiquitously present in humans. It regulates intracellular pH by removing an intracellular proton in exchange for an extracellular sodium. It consists of a 500 amino acid membrane domain plus a 315 amino acid, regulatory cytosolic tail. Here, we investigated the effect of mutation of two amino acids of the regulatory tail, Ser785 and Ser787, that were similar in location and context to two amino acids of the Arabidopsis Na+/H+ exchanger SOS1. Mutation of these two amino acids to either Ala or phosphomimetic Glu did not affect surface targeting but led to a slight reduction in the level of protein expressed. The activity of the NHE1 protein was reduced in the phosphomimetic mutations and the effect was due to a decrease in Vmax activity. The Ser to Glu mutations also caused a change in the apparent molecular weight of both the full-length protein and of the cytosolic tail of NHE1. A conformational change in this region was indicated by differential trypsin sensitivity. We also found that a peptide containing amino acids 783–790 bound to several more proximal regions of the NHE1 tail in in vitro protein interaction experiments. The results are the first characterization of these two amino acids and show that they have significant effects on enzyme kinetics and the structure of the NHE1 protein.


2018 ◽  
Vol 120 (12) ◽  
pp. 1321-1331 ◽  
Author(s):  
Hongnan Liu ◽  
Bie Tan ◽  
Bo Huang ◽  
Jianjun Li ◽  
Jing Wang ◽  
...  

AbstractCa2+-sensing receptor (CaSR) represents a potential therapeutic target for inflammatory bowel diseases and strongly prefers aromatic amino acid ligands. We investigated the regulatory effects of dietary supplementation with aromatic amino acids – tryptophan, phenylalanine and tyrosine (TPT) – on the CaSR signalling pathway and intestinal inflammatory response. The in vivo study was conducted with weanling piglets using a 2 × 2 factorial arrangement in a randomised complete block design. Piglets were fed a basal diet or a basal diet supplemented with TPT and with or without inflammatory challenge. The in vitro study was performed in porcine intestinal epithelial cell line to investigate the effects of TPT on inflammatory response using NPS-2143 to inhibit CaSR. Dietary supplementation of TPT alleviated histopathological injury and decreased myeloperoxidase activity in intestine challenged with lipopolysaccharide. Dietary supplementation of TPT decreased serum concentration of pro-inflammatory cytokines (IL-1β, IL-6, IL-8, IL-12, granulocyte-macrophage colony-stimulating factor, TNF-α), as well as the mRNA abundances of pro-inflammatory cytokines in intestine but enhanced anti-inflammatory cytokines IL-4 and transforming growth factor-β mRNA levels compared with pigs fed control diet and infected by lipopolysaccharide. Supplementation of TPT increased CaSR and phospholipase Cβ2 protein levels, but decreased inhibitor of NF-κB kinase α/β and inhibitor of NF-κB (IκB) protein levels in the lipopolysaccharide-challenged piglets. When the CaSR signalling pathway was blocked by NPS-2143, supplementation of TPT decreased the CaSR protein level, but enhanced phosphorylated NF-κB and IκB levels in IPEC-J2 cells. To conclude, supplementation of aromatic amino acids alleviated intestinal inflammation as mediated through the CaSR signalling pathway.


Food systems ◽  
2021 ◽  
Vol 4 (1) ◽  
pp. 4-11
Author(s):  
S. V. Zverev ◽  
V. I. Karpov ◽  
M. A. Nikitina

The paper emphasizes the importance of not only the quantitative but also qualitative composition of protein in nutrition. The authors propose protein classification into three main groups according to the concept of reference (ideal) protein. A mathematical model is examined to solve the task of rational mixture production upon the given profile of reference protein. Two variants of the criterion for formation of optimal composition are described. One of them presents the classical sum of squares of the residual for essential amino acid scores and 1. The second also presents the sum of squares of the residual for essential amino acid scores and 1 but with regard to only those amino acids, which scores are less than 1. The minima of these criteria at the set of variants for the content of ingredients are taken as targeted functions. The algorithm and the program of calculation were realized in the program environment Builder C++ 6.0. The macro flowchart of the algorithm is presented and detailed description of each block is given. The program interface before and after the start of the calculation module is shown. The main windows and interpretation of the presented data are described. An example of realization of the proposed mathematical apparatus when calculating a food model composition is given. Plant components (white kidney beans, flax, peanut, grit “Poltavskaya», dry red carrot) were used as an object of the research. Most plant proteins were incomplete. It is possible to regulate the chemical composition including correction of a protein profile by combination of plant raw materials. Analysis of alternative variants demonstrated that minimum essential amino acid score in the first composition was 0.79 (by the first criterion), in the second 1.0 (by the second criterion); the reference protein proportion in the mixture was 10.8 and 13.5, respectively, according to the first and second criterion. The comparative results by other quality indicators for protein in the mixture are also presented: the coefficient of amino acid score difference (CAASD), biological value (BV), coefficient of utility, essential amino acids index (IEAA).


2006 ◽  
Vol 84 (11) ◽  
pp. 1081-1095 ◽  
Author(s):  
Mackenzie E. Malo ◽  
Larry Fliegel

In mammalian eukaryotic cells, the Na+/H+ exchanger is a family of membrane proteins that regulates ions fluxes across membranes. Plasma membrane isoforms of this protein extrude 1 intracellular proton in exchange for 1 extracellular sodium. The family of Na+/H+ exchangers (NHEs) consists of 9 known isoforms, NHE1–NHE9. The NHE1 isoform was the first discovered, is the best characterized, and exists on the plasma membrane of all mammalian cells. It contains an N-terminal 500 amino acid membrane domain that transports ions, plus a 315 amino acid C-terminal, the intracellular regulatory domain. The Na+/H+ exchanger is regulated by both post-translational modifications including protein kinase-mediated phosphorylation, plus by a number of regulatory-binding proteins including phosphatidylinositol-4,5-bisphosphate, calcineurin homologous protein, ezrin, radixin and moesin, calmodulin, carbonic anhydrase II, and tescalcin. The Na+/H+ exchanger is involved in a variety of complex physiological and pathological events that include regulation of intracellular pH, cell movement, heart disease, and cancer. This review summarizes recent advances in the understanding of the physiological role and regulation of this protein.


2005 ◽  
Vol 81 (2) ◽  
pp. 257-264 ◽  
Author(s):  
H. L. Zhang ◽  
S. Y. Qiao ◽  
X. J. Chen ◽  
X. Wang ◽  
J. J. Xing ◽  
...  

AbstractThis experiment investigated the effects of feeding graded levels of a soya-bean protein product (HP300, Hamlet Protein A/S Company, Denmark) on endogenous ileal lysine loss, apparent ileal amino acid digestibility, standardized true ileal amino acid digestibility determined using the protein-free (PF) method, and real ileal amino acid digestibility determined using the homoarginine (HA) method. The soya-bean protein product was obtained by purifying and defattening soya bean via a proprietary microbial process that decreased the level of trypsin inhibitors and other anti-nutritional factors in soya bean. Six barrows, with an initial body weight of 37·4 ± 1·3 kg, were surgically fitted with simple T-cannulae at the distal ileum and offered six maize-starch-based diets according to a 6 × 6 Latin-square design. The six diets were formulated to provide 0, 50, 100, 150, 200, or 250 g crude protein (CP) per kg by dietary inclusion of 0, 90, 182, 274, 367 or 460 g/kg of soya-bean protein. Five kg of soya-bean protein product was guanidinated in order to estimate endogenous amino acid flow and real ileal amino acid digestibility. Chromium III oxide (5 g/kg) was included in the non-guanidinated diets while dysprosium chloride (0·1 g/kg) was included in the guanidinated diets as an indigestible marker. The experimental periods lasted 8 days. On day 6 of each period, ileal digesta was collected for 24 h to determine apparent and standardized true ileal amino acid digestibility of the non-guanidinated diets. At 08:00 h on day 8, the pigs were given a single meal of the diets containing guanidinated protein and their ileal digesta was collected for 24 h in order to determine the total HA flow and the real ileal digestibility of lysine. Endogenous ileal lysine flow appeared to follow a sigmoid curve starting at about 370 mg/kg dry matter (DM) intake for pigs given the PF diet and continuing asymptotically to about 750 mg/kg DM intake when the inclusion level of the soya-bean protein product was increased to 182 g/kg (100 g/kg of CP). The endogenous ileal lysine flow for pigs given the PF diet was similar (P > 0·05) to that of pigs given 90 g/kg soya-bean protein (50 g/kg of CP) and it increased sharply (P < 0·05) as the level of soya-bean protein increased from 90 to 182 g/kg (50 to 100 g/kg of CP). Thereafter, it was relatively constant (P > 0·05). With an increase in soya-bean protein, there was a quadratic increase (P < 0·01) in the apparent ileal digestibilities for all amino acids except valine and phenylalanine. Standardized true ileal amino acid digestibility decreased (P < 0·05) with an increase in soya-bean protein level. However, real ileal amino acid digestibilities were not influenced (P > 0·05) by soya-bean protein in the diet at levels between 90 and 367 g/kg (50 and 200 g/kg of CP). In conclusion, endogenous ileal lysine flow was not constant and was significantly affected by soya-bean protein level. The results of this study suggest that standardized true ileal amino acid digestibility should be measured between 100 and 200 g/kg of CP (182 and 367 g/kg soya-bean protein) while real ileal amino acid digestibility is unaffected by protein levels between 50 and 200 g/kg of CP (90 and 367 g/kg soya-bean protein).


1997 ◽  
Vol 78 (5) ◽  
pp. 823-831 ◽  
Author(s):  
Ana Triguero ◽  
Teresa Barber ◽  
Concha GarcÍa ◽  
Inmaculada R. Puertes ◽  
Juan Sastre ◽  
...  

To study the fate of l-cysteine and amino acid homeostasis in liver after the inhibition of the trans-sulfuration pathway, rats were treated with propargylglycine (PPG). At 4 h after the administration of PPG, liver cystathionase (EC 4.4.1.1) activity was undetectable, l-cystathionine levels were significantly higher, l-cysteine was unchanged and GSH concentration was significantly lower than values found in livers from control rats injected intraperitoneally with 0.15 M-NaCl. The hepatic levels of amino acids that are intermediates of the urea cycle, l-ornithine, l-citrulline and l-arginine and blood urea were significantly greater. Urea excretion was also higher in PPG-treated rats when compared with control rats. These data suggest a stimulation of ureagenesis in PPG-treated rats. The inhibition of γ-cystathionase was reflected in the blood levels of amino acids, because the L-methionine: l-cyst(e)ine ratio was significantly higher in PPG-treated rats than in control rats; blood concentration of cystathionine was also greater. Histological examination of liver and kidney showed no changes in PPG-treated rats when compared with controls. The administration of N-acetylcysteine (NAC) to PPG-treated rats reversed the changes in blood urea and in liver GSH. These data suggest that when liver l-cysteine production was impaired by the blockage of the trans-sulfuration pathway, the concentration of this amino acid was maintained mainly by an increase in protein degradation and by a depletion in GSH concentration that may spare l-cysteine.


2021 ◽  
Vol 99 (Supplement_1) ◽  
pp. 103-104
Author(s):  
Hanne Maribo

Abstract Diarrhoea in weaners has been commonly controlled by adding medicinal zinc (2500 ppm), but by June 2022 this was no longer allowed. In Denmark, antibiotics are accepted for therapeutic use only and usage is registered on pen level and is monitored by Danish authorities. This increases the risk of post-weaning diarrhoea. SEGES has tested several tools, additives e.g. organic acids, diet composition, raw materials e.g. blood plasma. Lowering the protein level in the diet post-weaning is very efficient, but adversely affects productivity. The latest results show on average that a reduction in protein from 19% to 15% in the weaner diet (6-9kg) results in a 60% reduction in diarrhoea; however, it also leads to a productivity loss of 1-1,5 euro. Reducing the protein level from 19% to 16,5% reduces the frequency of diarrhoea by 30% and the productivity loss by approx. 0,3 euro. A trial testing the possibility for compensation for this loss in the weaner period by adding extra protein and amino acids in the finisher diet (30–115 kg) is running now and preliminary results will be presented. Further results from trials reducing diarrhoea by reducing protein, a new way to calculate ideal protein and amino acid balances as well as results from concept tests with weaners will be presented. Further new results evaluating ideal protein and amino acid balances will be presented.


1993 ◽  
Vol 11 (2) ◽  
pp. 141-149 ◽  
Author(s):  
S M Duthie ◽  
P L Taylor ◽  
K A Eidne

ABSTRACT The cloning and characterization of the mouse TRH receptor (TRH-R) gene revealed an untranslated exon (exon 1), a single intron and an upstream dinucleotide repeat sequence (d(TG)16.d(AG)21) in the 5′ untranslated region (UTR). The coding region was contained almost entirely on a second exon (exon 2), with the final amino acid and stop codon at the COOH terminus of the gene encoded by a third exon (exon 3) flanked by two introns. The 3′ UTR was contained on the remainder of exon 3 and on the final exon (exon 4). Exon 3 (228 bp) corresponds exactly to a 228 bp deletion that exists in the rat TRH-R cDNA, but not in the mouse cDNA. The mouse TRH-R cDNA encodes a protein of 393 amino acids which is 96% homologous to the rat TRH-R protein of 412 amino acids, but is 19 amino acids shorter at its COOH terminus. The coding sequence for these 19 amino acids (plus 1 extra amino acid) does exist in the mouse TRH-R gene, but the sequence is encoded by exon 4, separated from the rest of the coding region by the stop codon and 223 bp of 3′ UTR on exon 3. Splicing of exon 3 in the mouse TRH-R gene would remove the last amino acid, the stop codon and the 223 bp of 3′ UTR, allowing transcription to continue into the 3′ UTR on exon 4, which encodes the 19 extra amino acids found in the rat cDNA. This would then result in an alternative 412 amino acid version of the mouse TRH-R protein, with 95% homology to the rat TRH-R. This study focused on the structural differences in the intracellular COOH-terminal tail of the receptor, which is known to be a functionally important domain in other members of the G protein-coupled receptor family. We have also recently characterized the human TRH-R cDNA, which revealed a third variant at the COOH terminus. Comparisons between mouse, rat and human TRH-Rs show that the amino acid sequences are virtually identical. However, significant differences between these species exist at the COOH terminus, with each TRH-R having a unique form of the COOH-terminal tail, beginning at exactly the same site and encoding 1, 20 and 6 amino acids in the mouse, rat and human respectively.


1985 ◽  
Vol 230 (1) ◽  
pp. 133-141 ◽  
Author(s):  
L P Chung ◽  
D R Bentley ◽  
K B Reid

By using synthetic oligonucleotides as probes, plasmid clones containing portions of cDNA coding for human C4b-binding protein were isolated from a liver cDNA library. The entire amino acid sequence of the C4b-binding protein can be predicted from this study of the cloned cDNA when allied to a previous sequence study at the protein level [Chung, Gagnon & Reid (1985) Mol. Immunol. 22, 427-435], in which over 55% of the amino acid sequence, including the N-terminal 62 residues, was obtained. The plasmid clones isolated allowed the unambiguous determination of 1717 nucleotides of cDNA sequence between the codon for the 32nd amino acid in the sequence of C4b-binding protein and the 164th nucleotide in the 3′ non-translated region. The sequence studies show that the secreted form of C4b-binding protein, found in plasma, is composed of chains of apparent Mr 70 000 that contains 549 amino acid residues. Examination of the protein and cDNA sequence results show that there are at least two polymorphic sites in the molecule. One is at position 44, which can be glutamine or threonine, and the other is at position 309, which can be tyrosine or histidine. Northern-blot analysis indicated that the mRNA for C4b-binding protein is approx. 2.5 kilobases long. The N-terminal 491 amino acids of C4b-binding protein can be divided into eight internal homologous regions, each approx. 60 amino acids long, which can be aligned by the presence in each region of four half-cystine, one tryptophan and several other conserved residues. These regions in C4b-binding protein are homologous with the three internal-homology regions that have been reported to be present within the Ba region of the complement enzyme factor B and also to the internal-homology regions found in the non-complement beta 2-glycoprotein I.


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