The Na+/H+ exchanger cytoplasmic tail: structure,function, and interactions with tescalcin

We characterized the regulatory cytoplasmic tail of the Na(+)/H(+) exchanger using a histidine-tagged protein containing the C-terminal 182 amino acids (His182). Both tescalcin and calmodulin, two Na(+)/H(+) exchanger binding proteins, bound to the His182 protein. Cascade blue was used to label the His182 protein. Calcium caused an increase in fluorescence, suggesting exposure of the label on the protein to a more hydrophilic environment. Decreasing external pH caused a transient increase in cascade blue fluorescence, followed by a decrease in fluorescence of the cascade blue labeled Na(+)/H(+) exchanger C-terminus. Tescalcin caused a decrease in fluorescence by labeled His182 protein, and calcium reversed this effect. Expression of tescalcin in vivo inhibited activity of the Na(+)/H(+) exchanger when there was an intact C-terminus of the protein. We examined the CD spectra of His182 in the presence and absence of tescalcin. The C-terminal amino acids demonstrated a very small amount of alpha-helical structure and much more beta-sheet and beta-turn. This was not greatly affected by the presence of tescalcin, but calcium caused an increase in the amount of beta-structure and a decrease in the unstructured proportion of the protein. Sedimentation equilibrium analysis demonstrated that the C-terminal 182 amino acids exist predominantly as a monomer. The results suggest that the C-terminus of the Na(+)/H(+) exchanger exists primarily as a monomeric protein that binds regulatory tescalcin and can change conformation depending on pH and calcium. Conformation changes in this region of the protein may be responsible for altering the pH sensitivity of the intact Na(+)/H(+) exchanger.     

Carbonic anhydrase II binds to and enhances activity of the Na+/H+ exchanger

We examined the ability of carbonic anhydrase II to bind to and affect the transport efficiency of the NHE1 isoform of the mammalian Na(+)/H(+) exchanger. The C-terminal region of NHE1 was expressed in Escherichia coli fused with an N-terminal glutathionine S-transferase or with a C-terminal polyhistidine tag. Using a microtiter plate binding assay we showed that the C-terminal region of NHE1 binds carbonic anhydrase II (CAII) and binding was stimulated by low pH and blocked by antibodies against the C-terminal of NHE1. The binding to NHE1 was confirmed by demonstrating protein-protein interaction using affinity blotting with CAII and immobilized NHE1 fusion proteins. CAII co-immunoprecipitated with NHE1 from CHO cells suggesting the proteins form a complex in vivo. In cells expressing CAII and NHE1, the H(+) transport rate was almost 2-fold greater than in cells expressing NHE1 alone. The CAII inhibitor acetazolamide significantly decreased the H(+) transport rate of NHE1 and transfection with a dominant negative CAII inhibited NHE1 activity. Phosphorylation of the C-terminal of NHE1 greatly increased the binding of CAII. Our study suggests that NHE1 transport efficiency is influenced by CAII, likely through a direct interaction at the C-terminal region. Regulation of NHE1 activity by phosphorylation could involve modulation of CAII binding.     

Properties of the Na+/H+ exchanger protein. Detergent-resistant aggregation and membrane microdistribution.

The Na+/H+ exchanger is a ubiquitous membrane protein of bacteria, plants and mammals. The first isoform discovered (NHE1) is present on the mammalian plasma membrane and transports one H+ out of cells in exchange for one extracellular Na+. With solubilization in standard SDS/PAGE buffer, this protein had a high tendency to aggregate when subjected to elevated temperature. The aggregates were stable and did not dissociate in high concentrations of SDS or 2-mercaptoethanol. We examined the distribution of the Na+/H+ exchanger within membrane subfractions. The Na+/H+ exchanger was found both in caveolin-containing fractions and, in lesser amounts, in higher density membrane fractions where the bulk of proteins were contained. Treatment with cytochalasin D caused only a minor reduction of the amount of Na+/H+ exchanger present in caveolin-enriched fractions suggesting an intact cytoskeleton was not important for NHE1 localization to these microdomains. Treatment of cells with methyl beta-cyclodextrin had a small stimulatory effect on Na+/H+ exchanger activity and reduced the amount of Na+/H+ exchanger in low density membrane fractions. Our study demonstrates that SDS cannot maintain the protein in a monomeric state suggesting that strong hydrophobic interactions are responsible for this temperature dependent aggregation behavior. In addition a large proportion of the Na+/H+ exchanger protein is found to be enriched in low density caveolin-containing fractions.     

(−)-Epigallocatechin Gallate Inhibits Asymmetric Dimethylarginine-Induced Injury in Human Brain Microvascular Endothelial Cells

(?)-Epigallocatechin gallate (EGCG) is the main polyphenol component of green tea (leaves of the Camellia sinensis plant). EGCG has been reported to protect human brain microvascular endothelial cells (HBMECs) against injury in several models. However, the exact mechanism is still unclear. In the current study we found that EGCG protected against asymmetric dimethylarginine (ADMA)-induced HBMEC injury, and inhibited ADMA-induced reactive oxygen species production and malondialdehyde expression. At the same time, we found that pretreatment with EGCG attenuated the upregulation of Bax and the downregulation of Bcl-2, thus confirming the cellular protective properties of EGCG against ADMA-induced apoptosis. Furthermore, we found that EGCG inhibited ADMA-induced phosphorylation of ERK1/2 and p-38, whose inhibitors relieved HBMEC injury. In conclusion, EGCG can protect against ADMA-induced HBMEC injury via the ERK1/2 and p38 MAPK pathways, which are involved in the underlying mechanisms of HBMEC injury in cerebral infarction.     

Diverse residues of intracellular loop 5 of the Na+/H+ exchanger modulate proton sensing,expression, activity and targeting

The mammalian Na⁺/H⁺ exchanger isoform 1 (NHE1) is an integral membrane protein that regulates intracellular pH (pH_i) by removing a single intracellular proton in exchange for one extracellular sodium ion. It is involved in cardiac hypertrophy and ischemia reperfusion damage to the heart and elevation of its activity is a trigger for breast cancer metastasis. NHE1 has an extensive 500 amino acid N-terminal membrane domain that mediates transport and consists of 12 transmembrane segments connected by intracellular and extracellular loops. Intracellular loops are hypothesized to modulate the sensitivity to pH_i. In this study, we characterized the structure and function of intracellular loop 5 (IL5), specifically amino acids 431–443. Mutation of eleven residues to alanine caused partial or nearly complete inhibition of transport; notably, mutation of residues L432, T433, I436, N437, R440 and K443 demonstrated these residues had critical roles in NHE1 function independent of effects on targeting or expression. The nuclear magnetic resonance (NMR) solution spectra of the IL5 peptide in a membrane mimetic sodium dodecyl sulfate solution revealed that IL5 has a stable three-dimensional structure with substantial alpha helical character. NMR chemical shifts indicated that K438 was in close proximity with W434. Overall, our results show that IL5 is a critical, intracellular loop with a propensity to form an alpha helix, and many residues of this intracellular loop are critical to proton sensing and ion transport.     

Induction of endoplasmic reticulum stress-induced beta-cell apoptosis and accumulation of polyubiquitinated proteins by human islet amyloid polypeptide

The islet in type 2 diabetes is characterized by an approximately 60% beta-cell deficit, increased beta-cell apoptosis, and islet amyloid derived from islet amyloid polypeptide (IAPP). Human IAPP (hIAPP) but not rodent IAPP (rIAPP) forms toxic oligomers and amyloid fibrils in an aqueous environment. We previously reported that overexpression of hIAPP in transgenic rats triggered endoplasmic reticulum (ER) stress-induced apoptosis in beta-cells. In the present study, we sought to establish whether the cytotoxic effects of hIAPP depend on its propensity to oligomerize, rather than as a consequence of protein overexpression. To accomplish this, we established a novel homozygous mouse model overexpressing rIAPP at a comparable expression rate and, on the same background, as a homozygous transgenic hIAPP mouse model previously reported to develop diabetes associated with beta-cell loss. We report that by 10 wk of age hIAPP mice develop diabetes with a deficit in beta-cell mass due to increased beta-cell apoptosis. The rIAPP transgenic mice counterparts do not develop diabetes or have decreased beta-cell mass. Both rIAPP and hIAPP transgenic mice have increased expression of BiP, but only hIAPP transgenic mice have elevated ER stress markers (X-box-binding protein-1, nuclear localized CCAAT/enhancer binding-protein homologous protein, active caspase-12, and accumulation of ubiquitinated proteins). These findings indicate that the beta-cell toxic effects of hIAPP depend on the propensity of IAPP to aggregate, but not on the consequence of protein overexpression.     

评价量规——主动学习的评价工具

新课程倡导学生主动学习,评价量规是对此学习方式进行教学和评价的有力工具。通过实例介绍评价量规的设计和应用,帮助教师理解评价量规,以期有更多的教师掌握和运用这一工具。     

滁菊的HPLC指纹图谱制作及其化学计量学分析

建立滁菊的HPLC指纹图谱,结合化学计量学手段对不同滁菊的指纹图谱进行分析研究,以期为滁菊的质量控制和产地追溯提供依据。采用HPLC法建立指纹图谱,并用中药色谱指纹图谱相似度评价系统(2012版)和系统聚类、主成分分析2种化学计量学法对指纹图谱和特征峰进行分析。分析结果发现样品有27个共有峰,12个滁菊样品具有较高的相似度,杭菊与滁菊的相似度较差,聚类分析与主成分分析结果和相似度分析结果一致。将HPLC指纹图谱与化学计量学结合可对滁菊进行鉴别和质量评价,为其质量控制和追溯提供了理论参考。