Impact of Temperature Dependent Sampling Procedures in Proteomics and Peptidomics – A Characterization of the Liver and Pancreas Post Mortem Degradome

Little is known about the nature of post mortem degradation of proteins and peptides on a global level, the so-called degradome. This is especially true for nonneural tissues. Degradome properties in relation to sampling procedures on different tissues are of great importance for the studies of, for instance, post translational modifications and/or the establishment of clinical biobanks. Here, snap freezing of fresh (<2 min post mortem time) mouse liver and pancreas tissue is compared with rapid heat stabilization with regard to effects on the proteome (using two-dimensional differential in-gel electrophoresis) and peptidome (using label free liquid chromatography). We report several proteins and peptides that exhibit heightened degradation sensitivity, for instance superoxide dismutase in liver, and peptidyl-prolyl cis-trans isomerase and insulin C-peptides in pancreas. Tissue sampling based on snap freezing produces a greater amount of degradation products and lower levels of endogenous peptides than rapid heat stabilization. We also demonstrate that solely snap freezing related degradation can be attenuated by subsequent heat stabilization. We conclude that tissue sampling involving a rapid heat stabilization step is preferable to freezing with regard to proteomic and peptidomic sample quality.The evolving maturation of the field of proteomics has, in the same way as in genomics, highlighted the need of better sampling procedures and sample preparation methodologies to minimize the effect of post mortem alterations. The aspect of sample quality is not new in any way and is relevant in most biomedical fields but has only lately started to receive adequate attention. The main factors influencing sample quality is storage temperature of the body until tissue removal (foremost a problem in clinical settings and extraction of less accessible tissue samples from model organisms) and post mortem interval (PMI)¹ (1–3). Post mortem degradation in during PMI is a well known compromising problem when studying endogenous peptides (2, 3) and has also been proven to affect the results of polypeptide (here defined as proteins larger than 10 kDa) studies (3–8). PMI degradation has mainly been studied on human or mouse brain tissue, using two-dimensional electrophoresis (2-DE), SDS-PAGE, and immunoblotting (1, 3–12). There are also a few proteomic studies on muscle tissue degradation in livestock (13–16).We and others have previously explored the effect of focused microwave irradiation with regard to sample quality, demonstrating that this method is more reliable than snap freezing in liquid nitrogen, especially with regard to post-translational modification (PTM) stability (2, 3, 17–20). An alternative method based on cryostat dissection with subsequent heat treatment through boiling has also been reported to improve endogenous peptide sample quality (21). Besides focused microwave irradiation, which is specifically used for rodent brain tissue sampling, we have also demonstrated the efficiency of rapid heat stabilization through conductivity with regard to sample degradation (3, 22). Although somewhat constrained by its dependence on how quickly the tissue is harvested from the body, the latter procedure has the added advantage that it can be used on any type of tissue and species, fresh as well as frozen. This study will compare effects of sampling procedures on the liver and pancreas degradome following rapid heat stabilization, the more traditional snap freezing, or the combination of snap freezing with subsequent heat stabilization.To summarize, this study investigated the effects of post mortem degradation in pancreas and liver. Both tissues are well studied because of their multiple functions in the body and their involvement in different diseases such as diabetes or hepatocarcinoma. Pancreas is especially interesting in this context as it displays endocrine secretion of peptides, and exocrine secretion of digestive enzymes, the later making it a protease rich tissue. We used both two-dimensional difference in gel electrophoresis (2D-DIGE) and label free liquid chromatography mass spectrometry (LC-MS) based differential peptide display (2, 18), the later to better investigate changes in small molecular fragment that are not easily detectable by gel-based methods. 2D-DIGE is an unrivaled methodology to characterize alterations in isoform patterns, which is an important aspect considering that post-translational modifications (PTMs) such as phosphorylations are especially sensitive to post mortem influence within a few minutes PMI (3). The peptidomics approach has been used in several studies to point out early post mortem changes and protein degradation that tissue undergo following sampling and is therefore a well-suited method (3, 18, 22).     

The K+ battery-regulating Arabidopsis K+ channel AKT2 is under the control of multiple post-translational steps

Potassium (K⁺) is an important nutrient for plants. It serves as a cofactor of various enzymes and as the major inorganic solute maintaining plant cell turgor. In a recent study, an as yet unknown role of K⁺ in plant homeostasis was shown. It was demonstrated that K⁺ gradients in vascular tissues can serve as an energy source for phloem (re)loading processes and that the voltage-gated K⁺ channels of the AKT2-type play a unique role in this process. The AKT2 channel can be converted by phosphorylation of specific serine residues (S210 and S329) into a non-rectifying channel that allows a rapid efflux of K⁺ from the sieve element/companion cells (SE/CC) complex. The energy of this flux is used by other transporters for phloem (re)loading processes. Nonetheless, the results do indicate that post-translational modifications at S210 and S329 alone cannot explain AKT2 regulation. Here, we discuss the existence of multiple post-translational modification steps that work in concert to convert AKT2 from an inward-rectifying into a non-rectifying K⁺ channel.Key words: potassium, channel, potassium channel, AKT2, phloem (re)loading, post-translational modifications, potassium batteryPotassium (K⁺) is the most abundant mineral element in plants, and together with nitrogen and phosphorous, is limiting for plant production in many natural and agricultural habitats. Voltage-gated K⁺ channels are key players in the acquisition of K⁺ ions from the soil and in its redistribution within the plant.¹ Structurally, these channels result from the assembly of four so-called α-subunits. The subunits are encoded by nine genes in Arabidopsis and both homo- and hetero-tetramers are expressed.^2,3 The K⁺ channel α-subunits can be categorized into four different subfamilies, based on the voltage-gating characteristics of the exogenous K⁺ conductance when expressed in an appropriate heterologous expression system. K_in α-subunits form hyperpolarization-activated channels that mediate K⁺ uptake.^4–7 K_out α-subunits form depolarization-activated channels that mediate K⁺ release from cells.^8–10 K_silent subunits appear unable to yield functional homomeric channels, but can combine with K_in subunits and fine-tune the K⁺-uptake properties of the resulting heteromeric channels.^11–14 Finally, K_weak α-subunits form channels with complex voltage-gating; they allow both K⁺ uptake and release.^15–19 In Arabidopsis, a single member is found in this subfamily, AKT2, and this channel can assemble in heteromeric channels with the K_in subunit KAT2.²⁰To date, only scarce and speculative information has been obtained for the function of K_weak channels. When expressed in heterologous expression systems, two different subpopulations of AKT2 channels differing in their sensitivity to voltage were found.²¹ Channels of the first type showed gating properties and currents analogous to that of K_in channels, while the other sort enabled a non-rectified (leak-like) current; they were open over the entire physiological voltage range.A given channel can be converted from one type to the other by post-translational modifications.²¹ A voltage-dependent phosphorylation was found to be an essential step for this switch,^22,23 although the kinase responsible for this conversion still needs to be uncovered.²⁴ In biophysical studies, mutant versions of the Arabidopsis K_weak channel subunit AKT2 have been created that showed impaired gating mode settings.^22,23 Recently, Gajdanowicz et al. generated transgenic Arabidopsis thaliana plants that express these mutant AKT2 channels in the background of the akt2-1 null-allele plant.²⁵ The major conclusion from analyses of these mutants is that the status switching of AKT2 from an inward-rectifying to a non-rectifying channel is crucial for plants to overcome energy-limiting conditions. This function of AKT2 could be correlated to its expression in phloem tissues. Selective expression of AKT2 under the control of the phloem companion cell-specific AtSUC2 promoter rescued the akt2-1 line, but conversely, selective expression of AKT2 under the control of the guard cell-specific GC1 promoter,²⁶ resulted in further impairment of plant growth (Fig. 1). By combining diverse experimental approaches with mathematical simulation methods, an existing model for phloem (re)loading^18,27 was fundamentally improved. This allowed the uncovering of a novel and interesting role of K⁺ in phloem physiology: K⁺ gradients present between the sieve element/companion cell (SE/CC) complex and the apoplast can serve as an energy source in phloem (re)loading processes. This “potassium battery” can be tapped by means of AKT2 regulation. This clarifies the observation of Deeken et al.²⁸ that in AKT2 loss-of-function mutant plants, assimilates leaking away from the sieve tube were not efficiently reloaded into the main phloem stream.Open in a separate windowFigure 1AKT2 expressed only in guard cells delays plant development. (A–C) Representative wild-type, akt2-1 and akt2-1+pGC1:AKT2 complementation plants grown for 7 weeks (A), 9 weeks (B) and 12 weeks (C) under 12-h day/12-h night conditions at normal light intensity (150 µmol m⁻² s⁻¹). (D) akt2-1+pGC1:AKT2 developed a similar number of leaves as the akt2-1 knock out plants, but bolting-time was delayed. (B and E) After 9 weeks, wild-type plants were at an advanced bolting stage, akt2-1 plants had started bolting, but only initial signs of bolting were visible in akt2-1+pGC1:AKT2 plants. (C and F) At 12 weeks, akt2-1 plants had caught up with the wild-type and akt2-1+pGC1:AKT2 was just starting to bolt, although rosette-leaves were showing clear signs of senescence. For the generation of akt2-1+pGC1:AKT2, the AKT2 cDNA was fused to the guard cell-specific GC1 promoter²⁶ kindly provided by J.I. Schroeder, San Diego. The pGC1:AKT2 construct was cloned into pGreen0229-35S by replacing the 35S promoter and then transformed into the akt2-1 knockout plant. All seeds were cold-treated for 24 h at 4°C. Plants were grown on artificial substrate (type GS-90, Einheitserde). After 2 weeks, seedlings were transferred to single pots. Plants were grown in 60% relative humidity at 21°C during the day and 18°C at night. Phenotypical analyses were done in the middle of the day. Data are shown as means ± SD of n ≥ 9 plants. Statistical analyses using Student''s t test: (D, WT/akt2-1: p < 2e-08; D, WT/pGC-AKT2: p < 2e-08; D, akt2-1/pGC-AKT2: p < 5e-03; E, WT/akt2-1: p < 4e-06; E, WT/pGC-AKT2: p < 1e-10; E, akt2-1/pGC-AKT2: p < 5e-04; F, WT/akt2-1: p = 0.51; F, WT/pGC-AKT2: p < 1e-10; F, akt2-1/pGC-AKT2: p < 1e-10).AKT2 expression is especially abundant in phloem tissues and the root stele, both of which are characterized by a poor availability of oxygen.^29,30 This local internal hypoxia impairs respiratory activity of the vascular tissue and concomitantly, respiratory ATP production is reduced.³¹ As a consequence, phloem transport is very susceptible to decreasing oxygen supply to the plant.^29,32 It is therefore comprehensible that the above mentioned support by the K⁺ driving force for sucrose retrieval is especially relevant in the phloem. Indeed Gajdanowicz et al.²⁵ showed that transgenic plants lacking the AKT2 K⁺ channel were severely impaired in growth when exposed to mild hypoxia (10% v:v), whereas growth of wild-type plants was unaffected by this treatment. These observations illustrate the importance of biochemical flexibility in plant cells to cope with the energetic consequences of the steep oxygen concentration gradients that generally occur in plant stems and roots.In fact, the role of K⁺ gradients in driving sugar, amino acid and organic acid transport across plant cell membranes was first suggested several decades ago.^33,34 Experimental evidence for this concept was provided by various tests in which pieces of plant tissue were incubated in solutions with different K⁺ concentrations and pH levels.^33,34 Unfortunately, at that time the lack of genetic information to support this hypothesis (e.g., identifying transporter proteins that could provide a molecular mechanism to explain the working mechanism of substrate transport driven by a K⁺-motive force) resulted in this idea falling into oblivion. Indeed, the unequivocal experimental observation of this new role of K⁺ gradients in phloem reloading is extremely challenging. Under normal experimental conditions, K⁺ fluxes and sucrose fluxes are coupled during phloem loading in source tissues and unloading in sink tissues. Nonetheless, computational simulations predict that under certain conditions, a local K⁺/Suc antiport is also thermodynamically possible. In this antiport system, the energy from the K⁺ gradient is used to transport Suc into the phloem. This process is only transient; flooding the apoplast with K⁺ will decrease the K⁺ gradient. However, the gradient can be maintained for longer if surrounding cells take up the apoplastic K⁺ for their own use. A K⁺/Suc antiport will not occur in obvious sink or source tissues since the energy balances in such cells are fundamentally different. Consequently, in these tissues only the coupled symport of K⁺ and Suc can be observed. However, the computational predictions allowed the identification of the experimental conditions under which the effect of the K⁺/Suc antiport system is empirically observable at the whole plant level.An essential role in the regulation of AKT2 is played by (de)phosphorylation events of serine residues at positions S210 and S329. The replacement of both serines by asparagine (AKT2-S210N-S329N) resulted in a K⁺-selective leak that is locked in a continuously open mode when the channels are expressed in Xenopus oocytes. Under certain conditions, plants expressing the AKT2-S210N-S329N mutation showed growth benefits over wild-type plants; akt2-1+AKT2-S210N-S329N plants reach the generative state faster, possess an increased number of leaves and increased fresh weight (Fig. 2). Intuitively, one would expect a continuously open channel to cause severe problems for the plant, not a benefit as was observed here. We therefore have to postulate that phosphorylation at residues AKT2-S210 and AKT2-S329 is insufficient for converting AKT2 from an inward-rectifying into a non-rectifying channel; other, as yet unknown mechanisms, must contribute to the switch in the AKT2 gating mode. Such a concept would correspond to results that would otherwise be hard to explain. For instance, when both serine residues were replaced by glutamate, the mutant AKT2-S210E-S329E still showed wild-type characteristics.²² The S to E substitution is expected to mimic the phosphorylated state better than the S to N replacement. Furthermore, position AKT2-K197 has a fundamental influence on the AKT2 gating mode.²³ AKT2 mutants with that particular lysine substituted with a serine are far less sensitive towards (de)phosphorylation; they display the characteristics of a pure inward-rectifying K⁺ channel,²³ and transgenic Arabidopsis plants expressing AKT2 channels with this substitution showed the characteristics of akt2-1 knock-out plants.²⁵ Initially, it was proposed that the positive charge is important for sensitizing AKT2 to phosphorylation. However, the charge-conserving mutant AKT2-K197R is similar to the charge inverting mutant AKT2-K197D,²³ a purely inward-rectifying channel (Fig. 3). We therefore need to take into account that in plants, K197 may also be a target of post-translational modification.³⁵ At present, we can explain the beneficial effect of the AKT2-S210N-S329N mutant on plant growth only by a multiple step regulation of AKT2 (Fig. 4). The double-N mutation would then bypass the phosphorylation step, but AKT2-S210N-S329N could still be deregulated into an inward-rectifying channel. Thus, AKT2 can be considered as a highly specialized K_in channel that can be converted into a leak-like channel by a cascade of post-translational modification steps.Open in a separate windowFigure 2Plants expressing the AKT2-S210N-S329N mutant reach the generative state faster than wild-type plants. The mutant channel AKT2-S210N-S329N was expressed under the control of the native AKT2 promoter in the akt2-1 knock-out background. (A) Photos of representative Arabidopsis thaliana plants grown 7 weeks under short day conditions (12-h day/12-h night, light intensity = 150 µE m⁻²s⁻¹). Seven weeks after sowing, plants expressing only AKT2-S210N-S329N mutant channels (n = 22) differed significantly (Student''s t test, p < 4e-05) from wild-type plants (n = 20) in the height of the main inflorescent stalk (B) and fresh weight (C). At later time points, these differences decrease.²⁵Open in a separate windowFigure 3The mutant AKT2-K197R channel is inward-rectifying. Steady-state current-voltage characteristics measured at the end of activation voltage steps. Currents were normalized to the current values measured at −145 mV in 10 mM K⁺ and are shown as means ± SD (n = 6).Open in a separate windowFigure 4Minimal model for AKT2 gating-mode regulation. To switch AKT2 from an inward-rectifying into a non-rectifying channel, at least two post-translational steps are postulated. (1) Phosphorylation at residues AKT2-S210 and AKT2-S329 (transitions [1]→[2] and [3]→[4]) and (2) a yet unknown modification that most likely involves the residue AKT2-K197 (transitions [1]→[3] and [2]→[4]). Only after both modifications will AKT2 allow the efflux of K⁺ (state [4]).     

Stimulation of ALK by the growth factor midkine renders glioma cells resistant to autophagy-mediated cell death

Δ?-tetrahydrocannabinol (THC), the main active component of marijuana, promotes cancer cell death via autophagy stimulation. We find that activation of the tyrosine kinase receptor ALK by its ligand midkine interferes with the signaling mechanism by which THC promotes autophagy-mediated glioma cell death.     

Cross-species transmission of a novel adenovirus associated with a fulminant pneumonia outbreak in a new world monkey colony

Adenoviruses are DNA viruses that naturally infect many vertebrates, including humans and monkeys, and cause a wide range of clinical illnesses in humans. Infection from individual strains has conventionally been thought to be species-specific. Here we applied the Virochip, a pan-viral microarray, to identify a novel adenovirus (TMAdV, titi monkey adenovirus) as the cause of a deadly outbreak in a closed colony of New World monkeys (titi monkeys; Callicebus cupreus) at the California National Primate Research Center (CNPRC). Among 65 titi monkeys housed in a building, 23 (34%) developed upper respiratory symptoms that progressed to fulminant pneumonia and hepatitis, and 19 of 23 monkeys, or 83% of those infected, died or were humanely euthanized. Whole-genome sequencing of TMAdV revealed that this adenovirus is a new species and highly divergent, sharing <57% pairwise nucleotide identity with other adenoviruses. Cultivation of TMAdV was successful in a human A549 lung adenocarcinoma cell line, but not in primary or established monkey kidney cells. At the onset of the outbreak, the researcher in closest contact with the monkeys developed an acute respiratory illness, with symptoms persisting for 4 weeks, and had a convalescent serum sample seropositive for TMAdV. A clinically ill family member, despite having no contact with the CNPRC, also tested positive, and screening of a set of 81 random adult blood donors from the Western United States detected TMAdV-specific neutralizing antibodies in 2 individuals (2/81, or 2.5%). These findings raise the possibility of zoonotic infection by TMAdV and human-to-human transmission of the virus in the population. Given the unusually high case fatality rate from the outbreak (83%), it is unlikely that titi monkeys are the native host species for TMAdV, and the natural reservoir of the virus is still unknown. The discovery of TMAdV, a novel adenovirus with the capacity to infect both monkeys and humans, suggests that adenoviruses should be monitored closely as potential causes of cross-species outbreaks.     

Isolation and identification of antiplasmodial N-alkylamides from Spilanthes acmella flowers using centrifugal partition chromatography and ESI-IT-TOF-MS

The development of new antiplasmodial drugs is of primary importance due to the growing problem of multi-drug resistance of malaria parasites. Spilanthes acmella, a plant traditionally used for the treatment of toothache, was targeted as a lead for its potential antiplasmodial activity. A systematic approach for investigating a suitable centrifugal partition chromatography (CPC) solvent system for N-alkylamides separation was reported. The partition behavior of three N-alkylamides has been studied using several biphasic solvent mixtures in search of an adequate CPC solvent system for this class of compounds. Major N-alkylamides in S. acmella were isolated from a methanolic crude extract of flowers by CPC with the solvent system heptanes-ethyl acetate-methanol-water (3:2:3:2, v/v/v/v). Four N-alkylamides were purified and the structures were illustrated by electrospray ionization-ion trap-time of flight-mass spectrometry (ESI-IT-TOF-MS), 1H nuclear magnetic resonance (1H NMR) and 13C nuclear magnetic resonance (13C NMR). The CPC fractions, which contained natural mixtures of phytochemicals, demonstrated significantly higher antiplasmodial activity compared to corresponding purified N-alkylamides, thus suggesting that interactions between these N-alkylamides may potentiate antiplasmodial bioactivity.     

Inter-domain communication mechanisms in an ABC importer: a molecular dynamics study of the MalFGK2E complex

ATP-Binding Cassette transporters are ubiquitous membrane proteins that convert the energy from ATP-binding and hydrolysis into conformational changes of the transmembrane region to allow the translocation of substrates against their concentration gradient. Despite the large amount of structural and biochemical data available for this family, it is still not clear how the energy obtained from ATP hydrolysis in the ATPase domains is "transmitted" to the transmembrane domains. In this work, we focus our attention on the consequences of hydrolysis and inorganic phosphate exit in the maltose uptake system (MalFGK(2)E) from Escherichia coli. The prime goal is to identify and map the structural changes occurring during an ATP-hydrolytic cycle. For that, we use extensive molecular dynamics simulations to study three potential intermediate states (with 10 replicates each): an ATP-bound, an ADP plus inorganic phosphate-bound and an ADP-bound state. Our results show that the residues presenting major rearrangements are located in the A-loop, in the helical sub-domain, and in the "EAA motif" (especially in the "coupling helices" region). Additionally, in one of the simulations with ADP we were able to observe the opening of the NBD dimer accompanied by the dissociation of ADP from the ABC signature motif, but not from its corresponding P-loop motif. This work, together with several other MD studies, suggests a common communication mechanism both for importers and exporters, in which ATP-hydrolysis induces conformational changes in the helical sub-domain region, in turn transferred to the transmembrane domains via the "coupling helices".