Molecular determinants of the hanatoxin binding in voltage-gated K+-channel drk1

The carboxyl terminus of S3 segment (S3(C)) in voltage-gated potassium channels was proposed to bear the binding site for gating modifier toxins like Hanatoxin and a helical secondary structural arrangement was suggested. Due to the lack of complete structure in high resolution for such a channel molecule, no further direct experimental data to elucidate the mechanism for their binding conformations could thus far be derived. In order to examine the putative three-dimensional structure of S3(C) and to illustrate the residues required for Hanatoxin binding, molecular simulation and docking were performed, based on the solution structure of Hanatoxin and the structural information from lysine-scanning results for S3(C) fragment. From our results, it is indicated that both hydrophobic and electrostatic interactions are utilized to stabilize the toxin binding. Detailed docking residues and appropriate orientation for binding regarding hydrophobic/-philic environments are also described. Compared with the functional data proposed by previous studies, the helical structural arrangement for the C-terminus of S3 segment in voltage-gated potassium channels can therefore be further emphasized.     

Low Potassium Dialysate as a Protective Factor of Sudden Cardiac Death in Hemodialysis Patients with Hyperkalemia

AimHyperkalemia increases the risk of sudden cardiac death (SCD) in hemodialysis patients. Our objective was to determine the association between administering low potassium dialysate to hyperkalemic hemodialysis patients and SCD.MethodsWe conducted a retrospective cohort study with patients undergoing maintenance hemodialysis from May 1, 2006, through December 31, 2013. The dialysate composition was adjusted over time according to monthly laboratory results. A 1.0 mEq/L potassium dialysate was applied in patients with predialysis hyperkalemia (>5.5 mEq/L) and was included as a time-dependent confounding factor. The clinical characteristics of enrolled patients, the incidence and timing of SCD and risk factors for all-cause mortality and SCD were analyzed.ResultsThere were 312 patients on maintenance hemodialysis during the study period. One hundred and fifty-seven patients had been dialyzed against a 1.0 mEq/L potassium dialysate at least once. The rates of all-cause mortality and SCD were 48.17 and 20.74 per 1000 patient-years, respectively. A 1.12-fold increase in the risk of SCD in the 24-hour period starting with the hemodialysis procedure and a 1.36-fold increase in the 24 hours preceding a weekly cycle were found (p = 0.017). Multivariate Cox proportional hazards models showed that age, diabetes mellitus and predialysis hyperkalemia (>5.0 mEq/L) were significant predictors of all-cause mortality and SCD. Exposure to 1.0 mEq/L potassium dialysate, Kt/V, and serum albumin were independent protective factors against all-cause mortality. Only exposure to 1.0 mEq/L potassium dialysate significantly prevented SCD (hazard ratio = 0.33, 95% CI = 0.13–0.85).ConclusionsUsing low potassium dialysate in hyperkalemic hemodialysis patients may prevent SCD.     

Involvement of the N- and C-terminal fragments of bovine pancreatic deoxyribonuclease in active protein folding

The three-dimensional structure of bovine pancreatic (bp) DNase revealed that its N- and C-termini form an antiparallel beta-sheet structure. The involvement of this beta-sheet structure in the active protein folding of bpDNase was thus investigated via a series of deletion and substitution variants. Several substitution variants of N-terminal Leu1 and C-terminal Leu259, and one variant with only the last Thr260 deleted, remained fully active. However, the other deletion variants, in which 2-10 amino acid residues were removed from the C- or N-terminus, all lost the DNase activity. The results indicated that the backbone hydrogen bonding in the antiparallel beta-sheet, rather than the side-chain interactions, is crucial for the correct protein folding. When the deletion variants were complemented with synthetic peptides of the deleted N- or C-terminal sequences, the DNase activity was generated. The highest DNase activity was generated when the C-terminal 10-residue-deleted brDNase(Delta251-260) was admixed with the C-terminal 10-residue peptide (peptide C10) in a molar ratio of 1:400. The noncovalent binding between brDNase(Delta251-260) and peptide C10 exhibited a dissociation constant of 48 microM. Circular dichroism spectra showed that the deletion variants were partially folded with mainly helical structures and that admixture with corresponding peptides facilitated their folding into the nativelike beta-sheet-rich structure. Thermal denaturation profiles also revealed that the transition temperature for brDNase(Delta251-260) was increased from 55 to 63 degrees C after incubation with peptide C10. The folding activation process for the deletion variant occurred in two stages, and Ca(2+) was required.     

CD44 fucosylation on mesenchymal stem cell enhances homing and macrophage polarization in ischemic kidney injury

The lack of homing ability possibly reduces the healing potential of bone-marrow-derived mesenchymal stem cells (MSCs). Therefore, transforming native CD44 on MSCs into a hematopoietic cell E-/L-selectin ligand (HCELL) that possesses potent E-selectin affinity might enhance the homing and regenerative abilities of MSCs. Through fucosyltransferase VI (FTVI) transfection, MSCs were fucosylated on N-glycans of CD44 to become HCELL positive, thus interacting with E-selectin on injured endothelial cells. HCELL expression facilitated MSC homing in kidneys within 24 h after injury and reduced lung stasis. An in vitro adhesion assay revealed that transfection enhanced the association between MSCs and hypoxic endothelial cells. In mice treated with HCELL-positive MSCs, the injured kidneys exhibited clusters of homing MSCs, whereas MSCs were rarely observed in mouse kidneys treated with HCELL-negative MSCs. Most MSCs were initially localized at the renal capsule, and some MSCs later migrated inward between tubules. Most homing MSCs were in close contact with inflammatory cells without tubular transdifferentiation. Furthermore, HCELL-positive MSCs substantially alleviated renal injury, partly by enhancing the polarization of infiltrating macrophages. In conclusion, engineering the glycan of CD44 on MSCs through FTVI transfection might enhance renotropism and the regenerating ability of MSCs in ischemic kidney injury.     

Erythropoietin suppresses epithelial to mesenchymal transition and intercepts Smad signal transduction through a MEK-dependent mechanism in pig kidney (LLC-PK1) cell lines

Purpose

Tumor growth factor-β1 (TGF-β1) plays a pivotal role in processes like kidney epithelial-mesenchymal transition (EMT) and interstitial fibrosis, which correlate well with progression of renal disease. Little is known about underlying mechanisms that regulate EMT. Based on the anatomical relationship between erythropoietin (EPO)-producing interstitial fibroblasts and adjacent tubular cells, we investigated the role of EPO in TGF-β1-mediated EMT and fibrosis in kidney injury.

Methods

We examined apoptosis and EMT in TGF-β1-treated LLC-PK1 cells in the presence or absence of EPO. We examined the effect of EPO on TGF-β1-mediated Smad signaling. Apoptosis and cell proliferation were assessed with flow cytometry and hemocytometry. We used Western blotting and indirect immunofluorescence to evaluate expression levels of TGF-β1 signal pathway proteins and EMT markers.

Results

We demonstrated that ZVAD-FMK (a caspase inhibitor) inhibited TGF-β1-induced apoptosis but did not inhibit EMT. In contrast, EPO reversed TGF-β1-mediated apoptosis and also partially inhibited TGF-β1-mediated EMT. We showed that EPO treatment suppressed TGF-β1-mediated signaling by inhibiting the phosphorylation and nuclear translocation of Smad 3. Inhibition of mitogen-activated protein kinase kinase 1 (MEK 1) either directly with PD98059 or with MEK 1 siRNA resulted in inhibition of EPO-mediated suppression of EMT and Smad signal transduction in TGF-β1-treated cells.

Conclusions

EPO inhibited apoptosis and EMT in TGF-β1-treated LLC-PK1 cells. This effect of EPO was partially mediated by a mitogen-activated protein kinase-dependent inhibition of Smad signal transduction.     

A smallest 6 kda metalloprotease,mini-matrilysin,in living world: a revolutionary conserved zinc-dependent proteolytic domain- helix-loop-helix catalytic zinc binding domain (ZBD)

Background

The Aim of this study is to study the minimum zinc dependent metalloprotease catalytic folding motif, helix B Met loop-helix C, with proteolytic catalytic activities in metzincin super family. The metzincin super family share a catalytic domain consisting of a twisted five-stranded β sheet and three long α helices (A, B and C). The catalytic zinc is at the bottom of the cleft and is ligated by three His residues in the consensus sequence motif, HEXXHXXGXXH, which is located in helix B and part of the adjacent Met turn region. An interesting question is - what is the minimum portion of the enzyme that still possesses catalytic and inhibitor recognition?”

Methods

We have expressed a 60-residue truncated form of matrilysin which retains only the helix B-Met turn-helix C region and deletes helix A and the five-stranded β sheet which form the upper portion of the active cleft. This is only 1/4 of the full catalytic domain. The E. coli derived 6 kDa MMP-7 ZBD fragments were purified and refolded. The proteolytic activities were analyzed by Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2 peptide assay and CM-transferrin zymography analysis. SC44463, BB94 and Phosphoramidon were computationally docked into the 3day structure of the human MMP7 ZBD and TAD and thermolysin using the docking program GOLD.

Results

This minimal 6 kDa matrilysin has been refolded and shown to have proteolytic activity in the Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2 peptide assay. Triton X-100 and heparin are important factors in the refolding environment for this mini-enzyme matrilysin. This minienzyme has the proteolytic activity towards peptide substrate, but the hexamer and octamer of the mini MMP-7 complex demonstrates the CM-transferrin proteolytic activities in zymographic analysis. Peptide digestion is inhibited by SC44463, specific MMP7 inhibitors, but not phosphorimadon. Interestingly, the mini MMP-7 can be processed by autolysis and producing ~ 6 ~ 7 kDa fragments. Thus, many of the functions of the enzyme are retained indicating that the helix B-Met loop-helix C is the minimal functional “domain” found to date for the matrixin family.

Conclusions

The helix B-Met loop-helix C folding conserved in metalloprotease metzincin super family is able to facilitate proteolytic catalysis for specific substrate and inhibitor recognition. The autolysis processing and producing 6 kDa mini MMP-7 is the smallest metalloprotease in living world.     

Involvement of a novel C-terminal kinase domain of Kir6.2 in the K-ATP channel rundown reactivation

Rundown is a generally encountered problem while recording K_ATP channel activity with inside-out patches. No assigned structural fragment related to this mechanism has yet been derived from any of the functional analyses performed. Therefore, based on a combined sequence and secondary structure alignment against known crystal structure of segments from closely related proteins, we propose here the three-dimensional structural model of an intracellular C-terminal domain of the Kir6.2 subunit in K_ATP channels. An E. coli CMP-kinase was suggested as template for the model building. The subdomain arrangement of this novel kinase domain and the structural correlation for UDP-docking are described. With structural-functional interpretation, we conclude that the reactivation of K_ATP channel rundown by MgATP or UDP is very possibly regulated by this intracellular kinase domain at the C-terminus of Kir6.2 subunit in K_ATP channels.     

Probing the catalytic mechanism of bovine pancreatic deoxyribonuclease I by chemical rescue

Previous structural and mutational studies of bovine pancreatic deoxyribonuclease I (bpDNase I) have demonstrated that the active site His134 and His252 played critical roles in catalysis. In our present study, mutations of these two His residues to Gln, Ala or Gly reduced the DNase activity by a factor of four to five orders of magnitude. When imidazole or primary amines were added exogenously to the Ala or Gly mutants, the residual DNase activities were substantially increased by 60-120-fold. The rescue with imidazole was pH- and concentration-dependent. The pH-activity profiles showed nearly bell-shaped curves, with the maximum activity enhancement for H134A at pH 6.0 and that for H252A at pH 7.5. These findings indicated that the protonated form of imidazole was responsible for the rescue in H134A, and the unprotonated form was for that in H252A, prompting us to assign unambiguously the roles for His134 as a general acid, and His252 as a general base, in bpDNase I catalysis.     

Putative secondary active site of bovine pancreatic deoxyribonuclease I

Previous structural studies based on the co-crystal of a complex between bovine pancreatic deoxyribonuclease I (bpDNase I) and a double-stranded DNA octamer d(GCGATCGC)(2) have suggested the presence of a putative secondary active site near Ser43. In our present study, several crucial amino acid residues postulated in this putative secondary active site, including Thr14, Ser43, and His44 were selected for site-directed mutagenesis. A series of single, double and triple mutants were thus constructed and tested for their DNase I activity by hyperchromicity assay. Substitution of each or both of Thr14 and Ser43 by alanine results in mutant enzymes retaining 30-70% of WT bpDNase I activity. However, when His44 was replaced by aspartic acid, either in the single, double, or triple mutant, the enzyme activities were drastically decreased to 0.5-5% that of WT bpDNase I. Interestingly, when cysteine was substituted for Thr14 or Ser43, the specific DNase activities of the mutant enzymes were substantially increased by 1.5-100-fold, comparing to their alanine substitution mutant counterparts. Two other more sensitive DNase activity assay method, plasmid scission and zymogram analyses further confirm these observations. These results suggested that His44 may play a critical role in substrate DNA binding in this putative secondary active site, and introduction of sulfhydryl groups at Thr14 and Ser43 may facilitate Mn(2+)-coordination and further contribute to the catalytic activity of bpDNase I.     

Functional characterization of the placental fusogenic membrane protein syncytin

Syncytin is an envelope protein of the human endogenous retrovirus family W (HERV-W). Syncytin is specifically expressed in the human placenta and mediates trophoblast cell fusion into the multinucleated syncytiotrophoblast layer. It is a polypeptide of 538 amino acids and is predicted to be posttranslationally cleaved into a surface (SU) subunit and a transmembrane (TM) subunit. Functional characterization of syncytin protein can aid understanding of the molecular mechanism underlying syncytin-mediated cell fusion. In this report, we studied the structure-function relationship of syncytin in 293T and HeLa cells transiently expressing wild-type syncytin or syncytin mutants generated by linker scanning and deletion mutagenesis. Of the 22 linker-inserted mutants, mutants InS51, InV139, InE156, InS493, InA506, and InL529 were fusogenic, suggesting that regions around amino acids S51, V139, and E156 in the SU subunit and S493, A506, and L529 in the cytoplasmic domain (CTM) of syncytin are flexible in conformation. Of the 17 deletion mutants, nine mutants with deletions in the region from amino acids 479 to 538 were fusogenic. The deletion mutant DelI480, containing only the first four amino acid residues in the cytoplasmic domain, had enhanced fusogenic activity in comparison with the wild-type. In addition, two heptad repeat regions (HRA and B) were defined in the TM subunit of syncytin. A peptide inhibitor derived from the C-terminal heptad repeat region (HRB) was shown to potently inhibit syncytin-mediated cell fusion. Our results suggest that the cytoplasmic domain of syncytin is not essential for syncytin-mediated fusion but may play a regulatory role, and an intramolecular interaction between HRA and B is involved in the fusion process.