We report the optimization of a novel redox-sensitive probe with enhanced dynamic range and an exceptionally well-positioned oxidative midpoint redox potential. The present work characterizes factors ...that contribute to the improved Förster resonance energy transfer (FRET) performance of this green fluorescent protein (GFP)-based redox sensor. The α-helical linker, which separates the FRET donor and acceptor, has been extended in the new probe and leads to a decreased FRET efficiency in the linker's reduced, 'FRET-off' state. Unexpectedly, the FRET efficiency is increased in the new linker's oxidized, 'FRET-on' state compared with the parent probe, in spite of the longer linker sequence. The combination of a lowered baseline 'FRET-off' and an increased 'FRET-on' signal significantly improves the dynamic range of the probe for a more robust discrimination of its reduced and oxidized linker states. Mutagenesis of the cysteine residues within the α-helix linker reveals the importance of the fourth, C-terminal cysteine and the relative insignificance of the second cysteine in forming the disulfide bridge to clamp the linker into the high-FRET, oxidized state. To further optimize the performance of the redox probe, various cyan fluorescent protein (CFP)/yellow fluorescent protein (YFP) FRET pairs, placed at opposite ends of the improved redox linker (RL7), were quantitatively compared and exchanged. We found that the CyPet/YPet and ECFP/YPet FRET pairs when attached to RL7 do not function well as sensitive redox probes due to a strong tendency to form heterodimers, which disrupt the α-helix. However, monomeric versions of CyPet and YPet (mCyPet and mYPet) eliminate dimerization and restore redox sensitivity of the probe. The best performing probe, ECFP-RL7-EYFP, exhibits an approximately six-fold increase in FRET efficiency in vitro when passing from the oxidized to the reduced state. We determined the midpoint redox potential of the probe to be -143 ± 6 mV, which is ideal for measuring glutathione (GSH/GSSG) redox potentials in oxidative compartments of mammalian cells (e.g. the endoplasmic reticulum).
Multiple drug resistance protein 4 (MRP4, ABCC4) belongs to the C subfamily of the ATP-binding cassette (ABC) transporter superfamily and participates in the transport of diverse antiviral and ...chemotherapeutic agents such as 6-mercaptopurine (6-MP) and 9-(2-phosphonyl methoxyethyl) adenine (PMEA). We have undertaken a comprehensive functional characterization of protein variants of MRP4 found in Caucasians and other ethnicities. A total of 11 MRP4 missense genetic variants (nonsynonymous SNPs), fused to green fluorescent protein (GFP), were examined in Xenopus laevis oocytes for their effect on expression, localization, and function of the transporter. Radiolabeled 6-MP and PMEA were chosen as transport substrates. All MRP4 protein variants were found to be expressed predominantly in the oocyte membrane. A total of four variants (Y556C, E757 K, V776I, and T1142 M) exhibited a 20% to 40% reduced expression level compared to the wild type. Efflux studies showed that 6-MP is transported by MRP4 in unmodified form. Compared to wild-type MRP4, the transmembrane variant V776I, revealed a significant lower activity in 6-MP transport, while the amino acid exchange Y556C in the WalkerB motif displayed significantly higher transport of PMEA. The transport properties of the other variants were comparable to wild-type MRP4. Our study shows that Xenopus oocytes are well suited to characterize MRP4 and its protein variants. Carriers of the rare MRP4 variants Y556C and V776I may have altered disposition of MRP4 substrates. Hum Mutat 29(5), 659-669, 2008.
Casein phosphopeptides (CPPs) obtained by enzymatic hydrolysis
in vitro of caseins, have been shown to enhance calcium solubility and to increase the calcification of embryonic rat bones in their ...diaphyseal area. Little is known about the direct effects of CPPs on cultured osteoblastic cells. Calcium in the microenvironment surrounding bone cells is not only important for the mineralization of the extracellular matrix, but it is believed to provide preosteblasts with a signal that modulates their proliferation and differentiation.
The aim of the present study was to investigate the direct effects of four selected casein phosphopeptides on osteoblastic cell (MC3T3-E1 cells) viability and differentiation. The selected peptides have been obtained by chemical synthesis and differed in the number of phosphorylated sites and in the amino acid spacing out two phosphorylated sites, in order to further characterize the relationship between structure and function.
The results obtained in this work demonstrated that CPPs may directly affect osteoblast-like cell growth, calcium uptake and ultimately calcium deposition in the extracellular matrix. The effects exerted by distinct CPPs on osteogenesis
in vitro can be either stimulatory or inhibitory. Differential short amino acid sequences in their molecules, like the -SpEE- and the -SpTSpEE-motifs, are likely the molecular determinants for their biological activities on osteoblastic cells. Moreover, two genetic variants of CPPs showing one amino acid change in their sequence may profoundly differ in their biological activities. Finally, our data may also suggest important clues about the role of intrinsic phosphorylated peptides derived from endogenous phosphorylated proteins in bone metabolism, apart from extrinsic CPPs.
Alternative splicing is a sophisticated nuclear process that regulates gene expression. It represents an important mechanism for enhancing the functional diversity of proteins. Our current knowledge ...of alternatively spliced variants is derived mainly from mRNA transcripts, and very little is known about their protein tertiary structures. We carried out a large-scale analysis of known alternatively spliced variants at both protein sequence and structure levels and have shown that threading is, in general, a viable approach for modeling structures of alternatively spliced variants. An examination of alternative splicing at the protein sequence level revealed that the size of splicing events follows the power law distribution and the majority of splicing isoforms harbor only one or two alternations. We examined alternative splicing in the context of protein 3D structures and found that the boundaries of alternative splicing events generally happen in coil regions of secondary structures and exposed residues and the majority of the sequences involved in splicing are located on the surface of proteins. In light of these findings, we then proceeded to demonstrate that threading represents a useful tool for structure prediction of alternative splicing isoforms and addressed the fold stability issue of threading-based structure prediction by molecular dynamics simulation. Our analysis and the insights gained have helped to establish a viable method for structure prediction of alternatively spliced isoforms at the genome scale.
Corynebacterium diphtheriae is the etiological agent of diphtheria, a potential fatal disease caused by a corynephage toxin. The expression of this diphtheria toxin is controlled via an ...iron‐dependent repressor with various functions (DtxR). Some mutations in the dtxR gene are associated with diminished activity or even with total loss of DtxR function. We conducted a molecular study to characterize the dtxR alleles harbored by 34 isolates of C. diphtheriae recovered from Romanian patients between 1961 and 2007. Three of the seven alleles identified in this study have not previously been described. Two new DtxR types were identified, one of which has an unusual polypeptide length. All the new DtxR types were found in toxigenic isolates, suggesting that they effectively regulate the expression of diphtheria toxin. Furthermore, one of the new DtxR identified was also found in a non‐toxigenic isolate, making it a potential source of toxigenic isolates after lysogenic conversion.
Abstract Background Participation of protein polymorphism is often considered in the pathogenesis of various diseases. Aberrant protein glycosylation has been recognized to play major roles in human ...disorders, including neurodegenerative diseases. Objective The aim of the study was to examine possible involvement of protein genetic variants and degree of glycosylation of some serum glycoproteins in the manifestation of neurodegenerative disorders in a Czech population sample. Methods Apolipoprotein (Apo) E and three main serum markers of glycosylation defects (transferrin, Tf, alpha1-antitrypsin, aAT and ApoCIII) in patients with Alzheimer's dementia (AD), Parkinson's disease (PD) and vascular dementia (n = 62, 139 and 44, respectively) were analyzed by isoelectric focusing. Children with serious neurological symptoms (n = 55) and three age-matched control groups (n = 45, 45 and 42) were examined for comparison. Results Of the supposedly pathognomonic protein variants Tf C2, aAT ZM and ApoE e4 only the latter was detected with higher frequency in AD patients; significant synergy of the C2/e4 allelic combination was not confirmed. The most prominent finding among PD adults was an increased appearance of Tf C2 allele and significant mean hypoglycosylation of ApoCIII, besides a C2/e4 positive correlation in PD seniors. Laboratory signs of Tf hypoglycosylation and a pattern of Tf/ApoCIII hyperglycosylation have been occasionally found.
BACKGROUND: The dietary guidelines targeted at reducing cardiovascular risk lead to largely heterogeneous responses in which genetic determinants are largely involved. OBJECTIVES: We evaluated the ...effect of fatty acid binding protein 2 (FABP2) Ala54Thr and microsomal triacylglycerol transfer protein (MTTP) -493G/T allelic variations on plasma lipid markers, at baseline and on the response to the 3-mo Medi-RIVAGE primary prevention study. DESIGN: Subjects with moderate cardiovascular disease risk (n = 169) were advised to reduce total and saturated dietary fats and to increase intake of monounsaturated and polyunsaturated fats. They were genotyped for FABP2 Ala54Thr and MTTP -493G/T allelic variations, and plasma was processed for cardiovascular risk marker analyses. RESULTS: At baseline, men and women homozygous for Thr54 presented a significant opposite profile for plasma oleic acid (18:1), triacylglycerol-rich lipoprotein (TRL) cholesterol, and TRL phospholipids. In addition, all Thr/Thr men presented higher 18:1 values than did women. For the MTTP -493G/T polymorphism, although all TT subjects presented high apolipoprotein B-48, a genotype x sex interaction was present for palmitic acid, linolenic acid, eicosatrienoic acid, and insulin. The prudent diet clearly improved plasma lipid markers. FABP2 genotype did not interact much with the amplitude of the response. However, for MTTP polymorphism, men homozygous for the T allele displayed a significantly more pronounced response than did men carrying the G allele, which is particularly evident by their larger decrease in the Framingham score. CONCLUSIONS: These 2 polymorphic loci are thus differently associated with the baseline lipid markers as well as with the response to nutritional recommendations, but both presented a marked sex-specific profile, with the response to diet being particularly efficient in men homozygous for the MTTP -493T allele.
Breast cancer is a frequently reported cancer in women all over the world. Several methods available to cure the breast cancer based on stage. This study focused on chemoprevention drugs of ...Aromatase, a potential target in breast cancer. Natural variants of Aromatase are very common; they have been collected and modeled, optimized the energy of mutated Aromatase protein. Reversible (Anastrozole) and irreversible (Exemestane) Aromatase inhibitors are selected and performed molecular docking studies of each drug against each variant to see the binding affinity impact on protein variant and drugs. In this comparative study, Anastrozole, a cumene derivative showed more binding affinity and Diethylstilbestrol showed weak binding affinity against among all drugs. The comparative molecular docking revealed that the binding affinity between drug and Aromatase protein variant is imprecise but fairly close; therefore the protein variants of Aromatase can be conceived to be equal for chemoprevention of breast cancer therapy.
This chapter describes the different methods used for localization of proteins and organelles in Pichia pastoris. A series of plasmids and a modified immunofluorescence protocol for localization and ...co-localization of proteins and organelles are described. Also included are protocols for the labeling of different subcellular organelles with vital stains.
Proteome analysis led to the identification and characterization of tumor‐associated protein variants by two‐dimensional electrophoresis and mass spectrometry. We focused on comparing the influence ...of genotoxic nitroso compounds N‐methyl‐N‐nitrosourea, diethylnitrosamine and N‐nitrosomorpholine and the nongenotoxic peroxisome proliferator Nafenopin as tumor‐inducing agents on the protein pattern of rat hepatomas. We found several tumor‐associated variants that represent members of the aldo‐keto reductase superfamily. Their induction and/or inhibition was specifically related to the carcinogen used for tumor induction. The most prominent tumor‐associated protein, rat aldose reductase‐like protein‐1 (rARLP‐1) (69% sequence identity to lens aldose reductase) and three additional types of rARLP‐1 were detected in nitroso compound‐induced rat hepatomas, while rat aldo‐keto reductase protein‐c (Rak‐c), a novel tumor‐associated variant (65% sequence identity with 3α‐hydroxysteroid dehydrogenase) was discovered in N‐methyl‐N‐nitrosourea‐induced hepatomas only. 3α‐Hydroxysteroid dehydrogenase and γ4‐3‐ketosteroid‐5β‐reductase, both liver‐specific enzymes, were reduced in amount in all hepatomas investigated, independent of their mode of induction. We conclude, that detoxification enzymes like 3α‐hydroxysteroid dehydrogenase (3α‐HSD) and γ4‐3‐ketosteroid‐5β‐reductase (5β‐Red) might be replaced in hepatomas by tumor‐associated proteins that are often present in the embryonal state, like the rARLPs or the Rak‐c protein. Their induction appears to reflect an altered constitutive pattern of detoxification enzymes, detoxifying toxic aldehydes being induced by nitroso compounds. In contrast, members of the aldo‐keto reductase superfamily have not been found in Nafenopin‐induced hepatomas. The pattern of tumor‐associated protein variants is apparently characteristic for a given group of initiating carcinogens. The hypothesis is proposed that carcinogens leave specific fingerprints at the proteome level of manifest liver tumors.
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