Nitric oxide, nitrosyl iron complexes, ferritin and frataxin: A well equipped team to preserve plant iron homeostasis

Iron is a key element in plant nutrition. Iron deficiency as well as iron overload results in serious metabolic disorders that affect photosynthesis, respiration and general plant fitness with direct consequences on crop production.More than 25% of the cultivable land possesses low iron availability due to high pH (calcareous soils). Plant biologists are challenged by this concern and aimed to find new avenues to ameliorate plant responses and keep iron homeostasis under control even at wide range of iron availability in various soils. For this purpose, detailed knowledge of iron uptake, transport, storage and interactions with cellular compounds will help to construct a more complete picture of its role as essential nutrient. In this review, we summarize and describe the recent findings involving four central players involved in keeping cellular iron homeostasis in plants: nitric oxide, ferritin, frataxin and nitrosyl iron complexes. We attempt to highlight the interactions among these actors in different scenarios occurring under iron deficiency or iron overload, and discuss their counteracting and/or coordinating actions leading to the control of iron homeostasis.     

Cloning and characterization of Chlorobium tepidum ferritin

The Chlorobium tepidum ferritin (CtFtn) gene was synthesized and cloned into a pET3a expression vector (Novagen). CtFtn was expressed in Escherichia coli and purified to electrophoretic homogeneity. Sequence analysis indicates that all the conserved amino acids required to form the Fe²⁺ oxidizing ferroxidase center are present. Ftn is highly conserved from bacteria to humans, each subunit folds into a 4-helical bundle (helices A-D), with a long loop connecting helices B and C, plus a fifth short E-helix at the C-terminus. Calculations based on the secondary structure of CtFtn predict that each of these helices forms. However, the sequence of CtFtn shows a much longer C-terminus with a significant number of polar amino acids. Size-exclusion chromatography shows that CtFtn elutes at a size consistent with a 24-subunit protein cage. Incubation of CtFtn with Fe²⁺ produced an increase in the absorbance at 310 nm consistent with the incorporation of iron inside CtFtn. Assays monitoring ferroxidase activity showed that CtFtn possesses ferroxidase activity but it is less active than human H-chain ferritin. Additionally, the iron loading capacity of CtFtn is significantly reduced compared to proteins from other organisms. We propose that the unique extended C-terminus in CtFtn causes the decreased iron loading in CtFtn and possibly influences the slower rate of iron oxidation at the ferroxidase center.     

Terminal fatty-acyl-CoA desaturase involved in sex pheromone biosynthesis in the winter moth (Operophtera brumata)

The winter moth (Operophtera brumata L., Lepidoptera: Geometridae) utilizes a single hydrocarbon, 1,Z3,Z6,Z9-nonadecatetraene, as its sex pheromone. We tested the hypothesis that a fatty acid precursor, Z11,Z14,Z17,19-nonadecanoic acid, is biosynthesized from ??-linolenic acid, through chain elongation by one 2-carbon unit, and subsequent methyl-terminus desaturation. Our results show that labeled ??-linolenic acid is indeed incorporated into the pheromone component in vivo. A fatty-acyl-CoA desaturase gene that we found to be expressed in the abdominal epidermal tissue, the presumed site of biosynthesis for type II pheromones, was characterized and expressed heterologously in a yeast system. The transgenic yeast expressing this insect derived gene could convert Z11,Z14,Z17-eicosatrienoic acid into Z11,Z14,Z17,19-eicosatetraenoic acid. These results provide evidence that a terminal desaturation step is involved in the winter moth pheromone biosynthesis, prior to the decarboxylation.     

Azido-iodo-N-benzyl derivatives of threo-methylphenidate (Ritalin, Concerta): Rational design, synthesis, pharmacological evaluation, and dopamine transporter photoaffinity labeling

In contrast to tropane-based compounds such as benztropine and cocaine, non-tropane-based photoaffinity ligands for the dopamine transporter (DAT) are relatively unexplored. Towards addressing this knowledge gap, ligands were synthesized in which the piperidine nitrogen of 3- and 4-iodomethylphenidate was substituted with a benzyl group bearing a photoreactive azide. Analog (±)-3a demonstrated modest DAT affinity and a radioiodinated version was shown to bind covalently to rat striatal DAT and hDAT expressed in cultured cells. Co-incubation of (±)-3a with nonradioactive d-(+)-methylphenidate or (−)-2-??-carbomethoxy-3-??-(4-fluorophenyl)tropane (??-CFT, WIN-35,428, a cocaine analog) blocked DAT labeling. Compound (±)-3a represents the first successful example of a DAT photoaffinity ligand based on the methylphenidate scaffold. Such ligands are expected to assist in mapping non-tropane ligand-binding pockets within plasma membrane monoamine transporters.     

Detection of 5-hydroxymethylcytosine in a combined glycosylation restriction analysis (CGRA) using restriction enzyme Taq(α)I

5-Hydroxymethylcytosine (5-hmC) is a newly discovered DNA base in mammalian cells that is believed to be another important epigenetic modification. Here we report the use of a methylation-insensitive restriction enzyme Taq^αI coupled with selective chemical labeling of 5-hmC in a combined glycosylation restriction analysis (CGRA) to detect 5-hmC in TCGA sequences. This method, differentiates fully versus hemi-hydroxymethylated cytosine in the CpG dinucleotide, adds a new tool to facilitate biological studies of 5-hmC.     

Rapidly photoactivatable ATP probes for specific labeling of tropomyosin within the actomyosin protein complex

Tropomyosin-specific photoaffinity adenosine triphosphate (ATP) probes have been first developed, in which a diazirine moiety is incorporated into the γ-phosphate group as a rapidly carbene-generating photophore. These probes clearly labeled tropomyosin in the presence of other actomyosin components, that is, myosin, actin, and troponins. The specific labeling of tropomyosin was easily identified by selective trapping of the photo-incorporated ATP probe on Fe³⁺-immobilized metal ion affinity chromatography (IMAC) beads. The characteristic nature of tropomyosin-specific photocross-linking was further confirmed with a biotin-carrying derivative of the ATP probe. These data suggest that the tropomyosin on the actin filament assembly is located in close proximity to the ATP binding cavity of myosin.     

Isolation,Purification and Labeling of Mouse Bone Marrow Neutrophils for Functional Studies and Adoptive Transfer Experiments

Neutrophils are critical effector cells of the innate immune system. They are rapidly recruited at sites of acute inflammation and exert protective or pathogenic effects depending on the inflammatory milieu. Nonetheless, despite the indispensable role of neutrophils in immunity, detailed understanding of the molecular factors that mediate neutrophils'' effector and immunopathogenic effects in different infectious diseases and inflammatory conditions is still lacking, partly because of their short half life, the difficulties with handling of these cells and the lack of reliable experimental protocols for obtaining sufficient numbers of neutrophils for downstream functional studies and adoptive transfer experiments. Therefore, simple, fast, economical and reliable methods are highly desirable for harvesting sufficient numbers of mouse neutrophils for assessing functions such as phagocytosis, killing, cytokine production, degranulation and trafficking. To that end, we present a reproducible density gradient centrifugation-based protocol, which can be adapted in any laboratory to isolate large numbers of neutrophils from the bone marrow of mice with high purity and viability. Moreover, we present a simple protocol that uses CellTracker dyes to label the isolated neutrophils, which can then be adoptively transferred into recipient mice and tracked in several tissues for at least 4 hr post-transfer using flow cytometry. Using this approach, differential labeling of neutrophils from wild-type and gene-deficient mice with different CellTracker dyes can be successfully employed to perform competitive repopulation studies for evaluating the direct role of specific genes in trafficking of neutrophils from the blood into target tissues in vivo .     

Characterizing monoclonal antibody structure by carboxyl group footprinting

Structural characterization of proteins and their antigen complexes is essential to the development of new biologic-based medicines. Amino acid-specific covalent labeling (CL) is well suited to probe such structures, especially for cases that are difficult to examine by alternative means due to size, complexity, or instability. We present here a detailed account of carboxyl group labeling (with glycine ethyl ester (GEE) tagging) applied to a glycosylated monoclonal antibody therapeutic (mAb). The experiments were optimized to preserve the structural integrity of the mAb, and experimental conditions were varied and replicated to establish the reproducibility of the technique. Homology-based models were generated and used to compare the solvent accessibility of the labeled residues, which include aspartic acid (D), glutamic acid (E), and the C-terminus (i.e., the target probes), with the experimental data in order to understand the accuracy of the approach. Data from the mAb were compared to reactivity measures of several model peptides to explain observed variations in reactivity. Attenuation of reactivity in otherwise solvent accessible probes is documented as arising from the effects of positive charge or bond formation between adjacent amine and carboxyl groups, the latter accompanied by observed water loss. A comparison of results with previously published data by Deperalta et al using hydroxyl radical footprinting showed that 55% (32/58) of target residues were GEE labeled in this study whereas the previous study reported 21% of the targets were labeled. Although the number of target residues in GEE labeling is fewer, the two approaches provide complementary information. The results highlight advantages of this approach, such as the ease of use at the bench top, the linearity of the dose response plots at high levels of labeling, reproducibility of replicate experiments (<2% variation in modification extent), the similar reactivity of the three target probes, and significant correlation of reactivity and solvent accessible surface area.     

Protease- and Acid-catalyzed Labeling Workflows Employing 18O-enriched Water

Stable isotopes are essential tools in biological mass spectrometry. Historically, ¹⁸O-stable isotopes have been extensively used to study the catalytic mechanisms of proteolytic enzymes^1-3. With the advent of mass spectrometry-based proteomics, the enzymatically-catalyzed incorporation of ¹⁸O-atoms from stable isotopically enriched water has become a popular method to quantitatively compare protein expression levels (reviewed by Fenselau and Yao⁴, Miyagi and Rao⁵ and Ye et al.⁶). ¹⁸O-labeling constitutes a simple and low-cost alternative to chemical (e.g. iTRAQ, ICAT) and metabolic (e.g. SILAC) labeling techniques⁷. Depending on the protease utilized, ¹⁸O-labeling can result in the incorporation of up to two ¹⁸O-atoms in the C-terminal carboxyl group of the cleavage product³. The labeling reaction can be subdivided into two independent processes, the peptide bond cleavage and the carboxyl oxygen exchange reaction⁸. In our PALeO (protease-assisted labeling employing ¹⁸O-enriched water) adaptation of enzymatic ¹⁸O-labeling, we utilized 50% ¹⁸O-enriched water to yield distinctive isotope signatures. In combination with high-resolution matrix-assisted laser desorption ionization time-of-flight tandem mass spectrometry (MALDI-TOF/TOF MS/MS), the characteristic isotope envelopes can be used to identify cleavage products with a high level of specificity. We previously have used the PALeO-methodology to detect and characterize endogenous proteases⁹ and monitor proteolytic reactions^10-11. Since PALeO encodes the very essence of the proteolytic cleavage reaction, the experimental setup is simple and biochemical enrichment steps of cleavage products can be circumvented. The PALeO-method can easily be extended to (i) time course experiments that monitor the dynamics of proteolytic cleavage reactions and (ii) the analysis of proteolysis in complex biological samples that represent physiological conditions. PALeO-TimeCourse experiments help identifying rate-limiting processing steps and reaction intermediates in complex proteolytic pathway reactions. Furthermore, the PALeO-reaction allows us to identify proteolytic enzymes such as the serine protease trypsin that is capable to rebind its cleavage products and catalyze the incorporation of a second ¹⁸O-atom. Such "double-labeling" enzymes can be used for postdigestion ¹⁸O-labeling, in which peptides are exclusively labeled by the carboxyl oxygen exchange reaction. Our third strategy extends labeling employing ¹⁸O-enriched water beyond enzymes and uses acidic pH conditions to introduce ¹⁸O-stable isotope signatures into peptides.     

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling processes. Extracting sterol-enriched membrane microdomains from plant cells for proteomic analysis is a difficult task mainly due to multiple preparation steps and sources for contaminations from other cellular compartments. The plasma membrane constitutes only about 5-20% of all the membranes in a plant cell, and therefore isolation of highly purified plasma membrane fraction is challenging. A frequently used method involves aqueous two-phase partitioning in polyethylene glycol and dextran, which yields plasma membrane vesicles with a purity of 95% ¹. Sterol-rich membrane microdomains within the plasma membrane are insoluble upon treatment with cold nonionic detergents at alkaline pH. This detergent-resistant membrane fraction can be separated from the bulk plasma membrane by ultracentrifugation in a sucrose gradient ². Subsequently, proteins can be extracted from the low density band of the sucrose gradient by methanol/chloroform precipitation. Extracted protein will then be trypsin digested, desalted and finally analyzed by LC-MS/MS. Our extraction protocol for sterol-rich microdomains is optimized for the preparation of clean detergent-resistant membrane fractions from Arabidopsis thaliana cell cultures.We use full metabolic labeling of Arabidopsis thaliana suspension cell cultures with K¹⁵NO₃ as the only nitrogen source for quantitative comparative proteomic studies following biological treatment of interest ³. By mixing equal ratios of labeled and unlabeled cell cultures for joint protein extraction the influence of preparation steps on final quantitative result is kept at a minimum. Also loss of material during extraction will affect both control and treatment samples in the same way, and therefore the ratio of light and heave peptide will remain constant. In the proposed method either labeled or unlabeled cell culture undergoes a biological treatment, while the other serves as control ⁴.