Human Polypeptide Research Articles

Substrate‐Guided Front‐Face Reaction Revealed by Combined Structural Snapshots and Metadynamics for the Polypeptide N‐Acetylgalactosaminyltransferase 2

AbstractThe retaining glycosyltransferase GalNAc‐T2 is a member of a large family of human polypeptide GalNAc‐transferases that is responsible for the post‐translational modification of many cell‐surface proteins. By the use of combined structural and computational approaches, we provide the first set of structural snapshots of the enzyme during the catalytic cycle and combine these with quantum‐mechanics/molecular‐mechanics (QM/MM) metadynamics to unravel the catalytic mechanism of this retaining enzyme at the atomic‐electronic level of detail. Our study provides a detailed structural rationale for an ordered bi–bi kinetic mechanism and reveals critical aspects of substrate recognition, which dictate the specificity for acceptor Thr versus Ser residues and enforce a front‐face SNi‐type reaction in which the substrate N‐acetyl sugar substituent coordinates efficient glycosyl transfer.

Culture Duration-, Donor-, and Medium-Dependent Changes in OATP1B3- Mediated Telmisartan Uptake in Human Hepatocytes

Human hepatic organic anion-transporting polypeptides (OATPs), including OATP1B1, 1B3, and 2B1, are expressed at the basolateral membrane of hepatocytes and mediate the uptake of a variety of compounds from blood into hepatocytes. The liver-specific OATPs are increasingly recognized as playing important roles in the pharmacokinetic (PK) of many drugs and thus, involved in the clinically significant drug-drug interactions (DDIs). However, the evaluation of the specific roles of individual OATPs in hepatocytes is challenging because of the lack of selective inhibitors and probe substrates for each OATP member. In the present study, the uptake activity of OATP1B3 was examined in human hepatocytes cultured up to 14 days using an in vitro uptake assay. The results showed that OATP-mediated uptake of rosuvastatin, a substrate for OATPs declined substantially in cultured human hepatocytes. In contrast, the uptake of OATP1B3-selective substrate telmisartan was not measureable at earlier culture periods, but became detectable on Day 7 and showed culture duration-dependent changes from Day 7 to 14. Quantitative polymerase chain reaction (qPCR) analyses illustrated that the OATP functional change was not correlated with messenger ribonucleic acid (mRNA) expression alteration in hepatocytes cultured for 3 hours or 7 days. The OATP1B3-mediated telmisartan uptake was also culture medium- and donor-dependent, and only observed in 3 of 5 lots of hepatocytes cultured in 2 of 3 media tested. These results show that using human hepatocytes cultured in certain conditions may provide an excellent addition to transfected cell lines as a way to distinguish OATP1B3 from other hepatic OATP family members, such as OATP1B1, to provide more understanding of OATP-mediated clinical DDI.

P0105 Inhibition of hepatocellular carcinoma growth by adenovirus-mediated expression of human telomerase reverse transcriptase COOH-27 terminal polypeptide in mice

Background A 27-kDa C-terminal fragment of human telomerase reverse transcriptase, hTERTC27, has previously been reported to inhibit the growth and tumorigenicity of HeLa human cervical cancer cells and U87-MG human glioblastoma multiforme cells. However, the antitumour effects of hTERTC27 in hepatoma and its underlying mechanisms are unclear. Methods In the current study, the therapeutic effect of hTERTC27, mediated by recombinant adenovirus, in hepatocellular carcinoma was explored in vitro and in vivo to investigate the possible mechanisms. Findings Recombinant adenovirus carrying hTERTC27 (rAdv-hTERTC27) effectively inhibited the growth and induced apoptosis of Hepa 1–6 cell lines. Dendritic cells transduced with rAdv-hTERTC27 were highly effective at inducing antigen-specific T cell proliferation and increasing the activated cytotoxicity of T cells against Hepa 1–6 cells. Hepatocellular carcinoma was inhibited significantly when a single dose of 5 × 10 7 pfu rAdv-hTERTC27 was administered intravenously. Interpretation Our results suggest that rAdv-hTERTC27 may serve as a reagent for intravenous administration when combined with telomerase-based gene therapy and immunotherapy for cancer.

Competing for the treasure in exceptions

Competing for the treasure in exceptions

Engineering of Sialylated Mucin-type O-Glycosylation in Plants

Proper N- and O-glycosylation of recombinant proteins is important for their biological function. Although the N-glycan processing pathway of different expression hosts has been successfully modified in the past, comparatively little attention has been paid to the generation of customized O-linked glycans. Plants are attractive hosts for engineering of O-glycosylation steps, as they contain no endogenous glycosyltransferases that perform mammalian-type Ser/Thr glycosylation and could interfere with the production of defined O-glycans. Here, we produced mucin-type O-GalNAc and core 1 O-linked glycan structures on recombinant human erythropoietin fused to an IgG heavy chain fragment (EPO-Fc) by transient expression in Nicotiana benthamiana plants. Furthermore, for the generation of sialylated core 1 structures constructs encoding human polypeptide:N-acetylgalactosaminyltransferase 2, Drosophila melanogaster core 1 β1,3-galactosyltransferase, human α2,3-sialyltransferase, and Mus musculus α2,6-sialyltransferase were transiently co-expressed in N. benthamiana together with EPO-Fc and the machinery for sialylation of N-glycans. The formation of significant amounts of mono- and disialylated O-linked glycans was confirmed by liquid chromatography-electrospray ionization-mass spectrometry. Analysis of the three EPO glycopeptides carrying N-glycans revealed the presence of biantennary structures with terminal sialic acid residues. Our data demonstrate that N. benthamiana plants are amenable to engineering of the O-glycosylation pathway and can produce well defined human-type O- and N-linked glycans on recombinant therapeutics.

The role of two natural flavonoids on human amylin aggregation

Misfolded amylin, the human polypeptide hormone, forms amyloid deposits in pancreatic islets. These amyloid deposits contribute to the dysfunction of beta-cells and the loss of beta-cell mass in type 2 diabetes mellitus (T2DM). Inhibition of amylin fibrillization has been regarded as a potential therapeutic approach for T2DM. Using i¬‚uorescence spectroscopic analysis with thioi¬‚avin T, the role of two naturally occurring flavonoids named myricetin and epigallocatechin gallate on human amylin hormone fibrillization and destabilization of fibrillar aggregates were examined under near physiological conditions. The results showed that after 168 h incubation by shaker incubator in 37°C. Myricetin at 10 and 40 µM repressed amylin amyloid formation by 25.3 and 22.4%, respectively (p<0.05), and the similar values of epigallocatechin gallate inhibited the formation of β-sheet structure by 18.1 and 16.7%, respectively (p<0.05). The obtained data also confirmed that amyloidal sheet opening was induced by myricetin and epigallocatechin gallate significantly (p<0.05). Therefore, it was concluded that islet amyloid cytotoxicity to β-cells may be reduced by these two flavonoids, and these compounds should be key molecules for the development of the therapeutics for diabetes mellitus. Key words: Human islet amyloid polypeptide, hyperglycemia, myricetin, epigallocatechin gallate, flavonoids.

Solution structure of the N-terminal domain of human polypeptide chain release factor eRF1

Solution structure of the N-terminal domain of human polypeptide chain release factor eRF1

Structure and dynamics in solution of the stop codon decoding N‐terminal domain of the human polypeptide chain release factor eRF1

The high-resolution NMR structure of the N-domain of human eRF1, responsible for stop codon recognition, has been determined in solution. The overall fold of the protein is the same as that found in the crystal structure. However, the structures of several loops, including those participating in stop codon decoding, are different. Analysis of the NMR relaxation data reveals that most of the regions with the highest structural discrepancy between the solution and solid states undergo internal motions on the ps-ns and ms time scales. The NMR data show that the N-domain of human eRF1 exists in two conformational states. The distribution of the residues having the largest chemical shift differences between the two forms indicates that helices α2 and α3, with the NIKS loop between them, can switch their orientation relative to the β-core of the protein. Such structural plasticity may be essential for stop codon recognition by human eRF1.

A Novel Monoclonal Antibody Against Cannabinoid Receptor 1

The cannabinoid receptor 1 (CBR1) is being widely investigated because of its specific structure and functions compared with other cannabinoid receptors. In this study, we immunized BALB/c mice with synthesized human CBR1 polypeptide and obtained a novel monoclonal antibody (MAb) against human CBR1. Analysis through enzyme-linked immunosorbent assay (ELISA), spot-ELISA, Western blot, and immunohistochemistry revealed that the MAb was specifically against recombinant human CBR1 protein, and its subtype and affinity constant (Kaff) were IgG2b/k and 7.85 × 10(8) M/L, respectively. Using this MAb we found that CBR1 is expressed on HL-7702 cells and lipid tissue, raising the possibility that the CBR1 may take a role in glucose and lipid metabolism. Thus, this antibody might facilitate studies for pathophysiology of diseases associated with glucose and lipid metabolism abnormality.

Engineering Mammalian Mucin-type O-Glycosylation in Plants

Mucin-type O-glycosylation is an important post-translational modification that confers a variety of biological properties and functions to proteins. This post-translational modification has a particularly complex and differentially regulated biosynthesis rendering prediction and control of where O-glycans are attached to proteins, and which structures are formed, difficult. Because plants are devoid of GalNAc-type O-glycosylation, we have assessed requirements for establishing human GalNAc O-glycosylation de novo in plants with the aim of developing cell systems with custom-designed O-glycosylation capacity. Transient expression of a Pseudomonas aeruginosa Glc(NAc) C4-epimerase and a human polypeptide GalNAc-transferase in leaves of Nicotiana benthamiana resulted in GalNAc O-glycosylation of co-expressed human O-glycoprotein substrates. A chimeric YFP construct containing a 3.5 tandem repeat sequence of MUC1 was glycosylated with up to three and five GalNAc residues when co-expressed with GalNAc-T2 and a combination of GalNAc-T2 and GalNAc-T4, respectively, as determined by mass spectrometry. O-Glycosylation was furthermore demonstrated on a tandem repeat of MUC16 and interferon α2b. In plants, prolines in certain classes of proteins are hydroxylated and further substituted with plant-specific O-glycosylation; unsubstituted hydroxyprolines were identified in our MUC1 construct. In summary, this study demonstrates that mammalian type O-glycosylation can be established in plants and that plants may serve as a host cell for production of recombinant O-glycoproteins with custom-designed O-glycosylation. The observed hydroxyproline modifications, however, call for additional future engineering efforts.

1H, 13C, and 15N chemical shifts assignments for human endothelial monocyte-activating polypeptide EMAP II

Endothelial and monocyte-activating polypeptide II (EMAP II) is a cytokine that plays an important role in inflammation, apoptosis and angiogenesis processes in tumour tissues. Structurally, the EMAP II is a 169 amino acid residues long C-terminal domain (residues 147-312) of auxiliary tRNA binding protein p43. In spite of existence in pdb databank of two X-ray structures there are some important aspects of EMAP II cytokine function which are still not fully understood in detail. To obtain information about 3D structure and backbone dynamic processes in solution we perform structure evaluation of human EMAP II cytokine by NMR spectroscopy. The standard approach to sequence-specific backbone assignment using 3D NMR data sets was not successful in our studies and was supplemented by recently developed 4D NMR experiments with random sampling of evolution time space. Here we report the backbone and side chain (1)H, (13)C, and (15)N chemical shifts in solution for recombinant EMAP II cytokine together with secondary structure provided by TALOS+software.

Developmental expression and organisation of fibrinogen genes in the zebrafish

The zebrafish is a model organism for studying vertebrate development and many human diseases. Orthologues of the majority of human coagulation factors are present in zebrafish, including fibrinogen. As a first step towards using zebrafish to model human fibrinogen disorders, we cloned the zebrafish fibrinogen cDNAs and made in situ hybridisations and quantitative reverse transcription-polymerase chain reactions (qRT-PCR) to detect zebrafish fibrinogen mRNAs. Prior to liver development or blood flow we detected zebrafish fibrinogen expression in the embryonic yolk syncytial layer and then in the early cells of the developing liver. While human fibrinogen is encoded by a three-gene, 50 kilobase (kb) cluster on chromosome 4 ( FGB-FGA-FGG ), recent genome assemblies showed that the zebrafish fgg gene appears distanced from fga and fgb , which we confirmed by in situ hybridisation. The zebrafish fibrinogen Bβ and γ protein chains are conserved at over 50% of amino acid positions, compared to the human polypeptides. The zebrafish Aα chain is less conserved and its C-terminal region is nearly 200 amino acids shorter than human Aα. We generated transgenic zebrafish which express a green fluorescent protein reporter gene under the control of a 1.6 kb regulatory region from zebrafish fgg . Transgenic embryos showed strong fluorescence in the developing liver, mimicking endogenous fibrinogen expression. This regulatory sequence can now be used for overexpression of transgenes in zebrafish hepatocytes. Our study is a proof-of-concept step towards using zebrafish to model human disease linked to fibrinogen gene mutations.

A transgene-encoded cell surface polypeptide for selection, in vivo tracking, and ablation of engineered cells

AbstractAn unmet need in cell engineering is the availability of a single transgene encoded, functionally inert, human polypeptide that can serve multiple purposes, including ex vivo cell selection, in vivo cell tracking, and as a target for in vivo cell ablation. Here we describe a truncated human EGFR polypeptide (huEGFRt) that is devoid of extracellular N-terminal ligand binding domains and intracellular receptor tyrosine kinase activity but retains the native amino acid sequence, type I transmembrane cell surface localization, and a conformationally intact binding epitope for pharmaceutical-grade anti-EGFR monoclonal antibody, cetuximab (Erbitux). After lentiviral transduction of human T cells with vectors that coordinately express tumor-specific chimeric antigen receptors and huEGFRt, we show that huEGFRt serves as a highly efficient selection epitope for chimeric antigen receptor+ T cells using biotinylated cetuximab in conjunction with current good manufacturing practices (cGMP)-grade anti-biotin immunomagnetic microbeads. Moreover, huEGFRt provides a cell surface marker for in vivo tracking of adoptively transferred T cells using both flow cytometry and immunohistochemistry, and a target for cetuximab-mediated antibody-dependent cellular cytotoxicity and in vivo elimination. The versatility of huEGFRt and the availability of pharmaceutical-grade reagents for its clinical application denote huEGFRt as a significant new tool for cellular engineering.

The CAG Promoter is More Active than the CEF Promoter for the Expression of Transgenes in a Mouse ES Cell Line E14-Derived EB3 Cells

ABSTRACTMouse embryonic stem (ES) cells are useful for characterizing the functions of specific genes in pluripotency and differentiation, especially through genetic modifications. The activity of promoters for transgene expression was considered to vary in different transfected ES cell lines, and basic scientific knowledge regarding promoter activities in mouse ES cell lines will be very valuable. In this report, we compared the activities of viral and housekeeping gene promoters in a mouse ES E14-derived EB3 cells and somatic cells both in transient and stable expression systems. An immediate early promoter of human cytomegalovirus was potently inactivated in a stable expression system in EB3 cells, and it was active in EB3 cells in a transient expression system and in somatic cells both in transient and stable expression systems. In contrast, the CAG and CEF promoters, which are chicken β-actin gene- and human polypeptide elongation factor-1α gene-based promoters, respectively, were capable of efficient expression of transgenes not only in a transient expression system, but also in a stable expression system even in EB3 cells. However, it was noteworthy that the CAG promoter was more active than the CEF promoter. To the best of our knowledge, this is the first delineation of promoter activities in E14-derived mouse ES cells both in transient and stable expression systems.

Bactericidal and Hemolytic Activities of Synthetic Peptides Derived from Granulysin

Granulysin is a human polypeptide produced by cytolytic cells active against a broad range of microbes. Three peptides covering the regions 25-50 (Gr-1 and Gr-2) and 39-62 (Gr-3) of granulysin were synthesized, and their in vitro activity against Mycobacterium tuberculosis was evaluated. The most active peptide was Gr-1C, containing a disulphide bridge, with Minimal Inhibitory Concentration value of 10.1 microM. In concentrations of up to 50 microM, Gr-1 and Gr2 didn't exceed 30% of hemolysis.

The Ability of Rodent Islet Amyloid Polypeptide To Inhibit Amyloid Formation by Human Islet Amyloid Polypeptide Has Important Implications for the Mechanism of Amyloid Formation and the Design of Inhibitors

Islet amyloid polypeptide (IAPP) is a 37-residue polypeptide hormone that is responsible for islet amyloid formation in type II diabetes. Human IAPP is extremely amyloidogenic, while rat IAPP and mouse IAPP do not form amyloid in vitro or in vivo. Rat IAPP and mouse IAPP have identical primary sequences, but differ from the human polypeptide at six positions, five of which are localized between residues 20 and 29. The ability of rat IAPP to inhibit amyloid formation by human IAPP was tested, and the rat peptide was found to be an effective inhibitor. Thioflavin-T fluorescence-monitored kinetic experiments, transmission electron microscopy, and circular dichroism showed that rat IAPP lengthened the lag phase for amyloid formation by human IAPP, slowed the growth rate, reduced the amount of amyloid fibrils produced in a dose-dependent manner, and altered the morphology of the fibrils. The inhibition of human IAPP amyloid formation by rat IAPP can be rationalized by a model that postulates formation of an early helical intermediate during amyloid formation where the helical region is localized to the N-terminal region of IAPP. The model predicts that proline mutations in the putative helical region should lead to ineffective inhibitors as should mutations that alter the peptide-peptide interaction interface. We confirmed this by testing the ability of A13P and F15D point mutants of rat IAPP to inhibit amyloid formation by human IAPP. Both these mutants were noticeably less effective inhibitors than wild-type rat IAPP. The implications for inhibitor design are discussed.

GNB3 C825T Polymorphism and Elevated Blood Pressure

Unlike in Europeans and Africans, the relationship between the human guanine nucleotide binding beta polypeptide 3 (GNB3) C825T gene polymorphism (rs5443) and blood pressures is inconsistent in Asians. The aim of the study was to investigate whether the GNB3 genotype demonstrates different associations with resting blood pressure and body fatness across cardio/respiratory fitness (CRF) levels. A total of 727 Korean women aged 31-60 years (mean, 47.8+/-5.4 years) participated in the study. In subgroup analyses of the obese group, TT individuals had significantly higher values of body weight than CC and CT individuals (p=0.006 and p=0.006, respectively) and body mass index (BMI) (p=0.002 and p=0.011, respectively). TT and CT individuals also tended to have higher CRF values than CC individuals. Regression analyses showed that the association between GNB3 genotype and resting blood pressure remained significant after adjustment for age and menopause, but was not significant after additional adjustment for body fatness. In summary, the findings of this study suggest that body fatness and CRF might modify the GNB3-mediated genetic susceptibility to elevated resting blood pressures in middle-aged Korean women.

Latent Transforming Growth Factor-β-Binding Protein-4 Regulates Transforming Growth Factor-β1 Bioavailability for Activation by Fibrogenic Lung Fibroblasts in Response to Bleomycin

Recent evidence suggests that subsets of lung fibroblasts differentially contribute to fibrogenic progression. We have previously shown that a subset of rat lung fibroblasts with fibrogenic characteristics [Thy-1 (-) fibroblasts] responds to stimuli (bleomycin, interleukin-4, etc) with increased latent transforming growth factor (TGF)-beta activation, whereas non-fibrogenic Thy-1-expressing [Thy-1 (+)] fibroblasts do not. Activation of latent TGF-beta1 by interstitial lung fibroblasts is critical for fibrogenic responses. To better understand the susceptibility of fibrogenic fibroblasts to the stimulation of TGF-beta activation, we examined the role of latent TGF-beta-binding proteins (LTBPs), key regulators of TGF-beta bioavailability and activation, in TGF-beta1 activation by these fibroblasts. Treatment of fibroblasts with bleomycin up-regulated LTBP-4 mRNA, protein, and soluble LTBP-4-bound large latent TGF-beta1 complexes in Thy-1 (-) fibroblasts to significantly higher levels than in Thy-1 (+) fibroblasts. Bleomycin-induced TGF-beta1 activation required LTBP-4, since lung fibroblasts deficient in LTBP-4 did not activate TGF-beta1. Expression of LTBP-4 restored TGF-beta1 activation in response to bleomycin, but expression either of LTBP-4 lacking the TGF-beta-binding site or only the TGF-beta-binding domain did not. Bleomycin treatment of mice increased LTBP-4 expression in the lung. Thy-1 knockout mice had increased levels of both LTBP-4 expression and TGF-beta activation, as well as enhanced Smad3 phosphorylation compared with wild-type mice. Together, these data identify a critical role for LTBP-4 in the regulation of latent TGF-beta1 activation in bleomycin-induced lung fibrosis.

The functional consequences of genetic variations in transporter genes encoding human organic anion-transporting polypeptide family members

It is increasingly recognised that uptake transporters of the organic anion-transporting polypeptide (OATP) family play important roles in drug absorption, distribution and excretion. They are expressed in a variety of different tissues, including gut, brain, kidney and liver. Substrates of OATPs include several endogenous substances, such as bile salts and hormones, and drugs such as HMG-CoA reductase inhibitors (e.g., pravastatin), cytotoxic drugs and antibiotics. Recent advances in the pharmacogenetics of OATPs have demonstrated that variations (polymorphisms) in genes encoding human OATPs can explain parts of the interindividual variability in the pharmacokinetics of drugs and, thus, contribute to the interethnic and interindividual variability in drug response. This review focuses on consequences of these genetic variations and summarises in vivo as well as in vitro analyses demonstrating the impact of polymorphisms in genes encoding OATPs on transport and pharmacokinetics of drugs.

NMR assignments of the C-terminal domain of human polypeptide release factor eRF1

We report NMR assignments of the protein backbone of the C-terminal domain (163 a.a.) of human class 1 translation termination factor eRF1. It was found that several protein loop residues exist in two slowly interconverting conformational states.