Early Proteomic Changes Research Articles

Dysregulation of Metabolism and Proteostasis in Skeletal Muscle of a Presymptomatic Pompe Mouse Model

Pompe disease is a rare genetic metabolic disorder caused by mutations in acid-alpha glucoside (GAA) leading to pathological lysosomal glycogen accumulation associated with skeletal muscle weakness, respiratory difficulties and cardiomyopathy, dependent from the GAA residual enzyme activity. This study aimed to investigate early proteomic changes in a mouse model of Pompe disease and identify potential therapeutic pathways using proteomic analysis of skeletal muscles from pre-symptomatic Pompe mice. For this purpose, quadriceps samples of Gaa6neo/6neo mutant (Pompe) and wildtype mice, at the age of six weeks, were studied with three biological replicates for each group. The data were validated with skeletal muscle morphology, immunofluorescence studies and western blot analysis. Proteomic profiling identified 538 significantly upregulated and 16 significantly downregulated proteins in quadriceps muscles derived from Pompe animals compared to wildtype mice. The majority of significantly upregulated proteins were involved in metabolism, translation, folding, degrading and vesicular transport, with some having crucial roles in the etiopathology of other neurological or neuromuscular diseases. This study highlights the importance of the early diagnosis and treatment of Pompe disease and suggests potential add-on therapeutic strategies targeting protein dysregulations.

THRONCAT: metabolic labeling of newly synthesized proteins using a bioorthogonal threonine analog

Profiling the nascent cellular proteome and capturing early proteomic changes in response to external stimuli provides valuable insights into cellular physiology. Existing metabolic protein labeling approaches based on bioorthogonal methionine- or puromycin analogs allow for the selective visualization and enrichment of newly synthesized proteins. However, their applications are limited as they often require methionine-free conditions, auxotrophic cells and/or are toxic to cells. Here, we introduce THRONCAT, a threonine-derived non-canonical amino acid tagging method based on the bioorthogonal threonine analog β-ethynylserine (βES) that enables efficient labeling of the nascent proteome in complete growth media within minutes. We use THRONCAT for the visualization and enrichment of nascent proteins in bacteria, mammalian cells and Drosophila melanogaster. We profile immediate proteome dynamics of B-cells in response to B-cell receptor activation simply by adding βES to the culture medium, demonstrating the ease-of-use of the method and its potential to address diverse biological questions. In addition, using a Drosophila model of Charcot-Marie-Tooth peripheral neuropathy, we show that THRONCAT enables visualization and quantification of relative protein synthesis rates in specific cell types in vivo.

Adaptation and Changes in Actin Dynamics and Cell Motility as Early Responses of Cultured Mammalian Cells to Altered Gravitational Vector.

Cultured mammalian cells have been shown to respond to microgravity (μG), but the molecular mechanism is still unknown. The study we report here is focused on molecular and cellular events that occur within a short period of time, which may be related to gravity sensing by cells. Our assumption is that the gravity-sensing mechanism is activated as soon as cells are exposed to any new gravitational environment. To study the molecular events, we exposed cells to simulated μG (SμG) for 15 min, 30 min, 1 h, 2 h, 4 h, and 8 h using a three-dimensional clinostat and made cell lysates, which were then analyzed by reverse phase protein arrays (RPPAs) using a panel of 453 different antibodies. By comparing the RPPA data from cells cultured at 1G with those of cells under SμG, we identified a total of 35 proteomic changes in the SμG samples and found that 20 of these changes took place, mostly transiently, within 30 min. In the 4 h and 8 h samples, there were only two RPPA changes, suggesting that the physiology of these cells is practically indistinguishable from that of cells cultured at 1 G. Among the proteins involved in the early proteomic changes were those that regulate cell motility and cytoskeletal organization. To see whether changes in gravitational environment indeed activate cell motility, we flipped the culture dish upside down (directional change in gravity vector) and studied cell migration and actin cytoskeletal organization. We found that compared with cells grown right-side up, upside-down cells transiently lost stress fibers and rapidly developed lamellipodia, which was supported by increased activity of Ras-related C3 botulinum toxin substrate 1 (Rac1). The upside-down cells also increased their migratory activity. It is possible that these early molecular and cellular events play roles in gravity sensing by mammalian cells. Our study also indicated that these early responses are transient, suggesting that cells appear to adapt physiologically to a new gravitational environment.

The potential of remdesivir to affect function, metabolism and proliferation of cardiac and kidney cells in vitro

Remdesivir is a prodrug of a nucleoside analog and the first antiviral therapeutic approved for coronavirus disease. Recent cardiac safety concerns and reports on remdesivir-related acute kidney injury call for a better characterization of remdesivir toxicity and understanding of the underlying mechanisms. Here, we performed an in vitro toxicity assessment of remdesivir around clinically relevant concentrations (Cmax 9 µM) using H9c2 rat cardiomyoblasts, neonatal mouse cardiomyocytes (NMCM), rat NRK-52E and human RPTEC/TERT1 cells as cell models for the assessment of cardiotoxicity or nephrotoxicity, respectively. Due to the known potential of nucleoside analogs for the induction of mitochondrial toxicity, we assessed mitochondrial function in response to remdesivir treatment, early proteomic changes in NMCM and RPTEC/TERT1 cells and the contractile function of NMCM. Short-term treatments (24 h) of H9c2 and NRK-52E cells with remdesivir adversely affected cell viability by inhibition of proliferation as determined by significantly decreased 3H-thymidine uptake. Mitochondrial toxicity of remdesivir (1.6–3.1 µM) in cardiac cells was evident by a significant decrease in oxygen consumption, a collapse of mitochondrial membrane potential and an increase in lactate secretion after a 24–48-h treatment. This was supported by early proteomic changes of respiratory chain proteins and intermediate filaments that are typically involved in mitochondrial reorganization. Functionally, an impedance-based analysis showed that remdesivir (6.25 µM) affected the beat rate and contractility of NMCM. In conclusion, we identified adverse effects of remdesivir in cardiac and kidney cells at clinically relevant concentrations, suggesting a careful evaluation of therapeutic use in patients at risk for cardiovascular or kidney disease.

Molecular origin of somatostatin-positive neuron vulnerability.

Reduced somatostatin (SST) and dysfunction of SST-positive (SST+) neurons are hallmarks of neurological disorders and associated with mood disturbances, but the molecular origin of SST+ neuron vulnerability is unknown. Using chronic psychosocial stress as a paradigm to induce elevated behavioral emotionality in rodents, we report a selective vulnerability of SST+ neurons through exacerbated unfolded protein response (UPR) of the endoplasmic reticulum (ER), or ER stress, in the prefrontal cortex. We next show that genetically suppressing ER stress in SST+ neurons, but not in pyramidal neurons, normalized behavioral emotionality induced by psychosocial stress. In search for intrinsic factors mediating SST+ neuron vulnerability, we found that the forced expression of the SST precursor protein (preproSST) in SST+ neurons, mimicking psychosocial stress-induced early proteomic changes, induces ER stress, whereas mature SST or processing-incompetent preproSST does not. Biochemical analyses further show that psychosocial stress induces SST protein aggregation under elevated ER stress conditions. These results demonstrate that SST processing in the ER is a SST+ neuron-intrinsic vulnerability factor under conditions of sustained or over-activated UPR, hence negatively impacting SST+ neuron functions. Combined with observations in major medical illness, such as diabetes, where excess ER processing of preproinsulin similarly causes ER stress and β cell dysfunction, this suggests a universal mechanism for proteinopathy that is induced by excess processing of native endogenous proteins, playing critical pathophysiological roles that extend to neuropsychiatric disorders.

Proteomic characterization of early lung response to breast cancer metastasis in mice

IntroductionThe tumor-promoting rearrangement of the lungs facilitates the process of cancer cell survival in a foreign microenvironment and enables their protection against immune defense. The study aimed to define the fingerprint of the early rearrangement of the lungs via the proteomic profiling of the lung tissue in the experimental model of tumor metastasis in a murine 4T1 mammary adenocarcinoma. Materials and methodsThe studies were performed on 7-8-week-old BALB/c female mice. Viable 4T1 cancer cells were orthotopically inoculated into the right mammary fat pad. The experiment was performed in the early phase of the tumor metastasis one and two weeks after cancer cell inoculation. The comparative analysis of protein profiles was carried out with the aid of the two-dimensional difference in gel electrophoresis (2D-DIGE). Proteins, of which expression differed significantly, were identified using nano-liquid chromatography coupled to a high-resolution mass spectrometry (nanoLC/hybrid ion trap- Orbitrap XL Discovery). ResultsPalpable primary tumors were noted in the 2nd week after cancer cell inoculation. The investigated period preceded the formation of numerous macrometastases in the lungs, however the metastasis-promoting changes were visible very early. Primary tumor-induced inflammation developed in the lungs as early as after the 1st week and progressed during the 2nd week, accompanied by increased concentration of 2-OH-E+, an oxidative stress marker, and imbalance in nitric oxide metabolites, pointing to endothelium dysfunction. The early proteomic changes in the lungs in the 1st week after 4T1 cell inoculation resulted in the reorganization of lung tissue structure [actin, cytoplasmic 1 (Actb), tubulin beta chain (Tubb5), lamin-B1 (Lmnb1), serine protease inhibitor A3K (Serpina3k)] and activation of defense mechanisms [selenium-binding protein 1 (Selenbp1), endoplasmin (Hsp90b1), stress 70 protein, mitochondrial (Hspa9), heat shock protein HSP 90-beta (Hsp90ab1)], but also modifications in metabolic pathways [glucose-6-phosphate 1-dehydrogenase X (G6pdx), ATP synthase subunit beta, mitochondrial (Atp5b), L-lactate dehydrogenase B chain (Ldhb)]. Further development of the solid tumor after the 2nd week following cancer cell inoculation, secretion of prolific tumor-derived factors as well as the presence of the increasing number of circulating cancer cells and extravasation processes further impose reorganization of the lung tissue [Actb, vimentin (Vim), clathrin light chain A (Clta)], altering additional metabolic pathways [annexin A5 (Anxa5), Rho GDP-dissociation inhibitor 2 (Arhgdib), complement 1 Q subcomponent-binding protein, mitochondrial (C1qbp), 14-3-3 protein zeta/delta (Ywhaz), peroxiredoxin-6 (Prdx6), chitinase-like protein 4 (Chi3l4), reticulocalbin-1 (Rcn1), EF-hand domain-containing protein D2 (Efhd2), calumenin (Calu)]. Interestingly, many of differentially expressed proteins were involved in calcium homeostasis (Rcn1, Efhd2, Calu, Actb, Vim, Lmnb1, Clta, Tubb5, Serpina3k, Hsp90b1, Hsp90ab1, Hspa9. G6pdx, Atp5b, Anxa5, Arhgdib, Ywhaz). ConclusionThe analysis enabled revealing the importance of calcium signaling during the early phase of metastasis development, early cytoskeleton and extracellular matrix reorganization, activation of defense mechanisms and metabolic adaptations. It seems that the tissue response is an interplay between pro- and anti-metastatic mechanisms accompanied by inflammation, oxidative stress and dysfunction of the barrier endothelial cells.

COMPARATIVE PROTEOMIC ANALYSIS IN MICRODISSECTED RENAL VESSELS OF HYPERTENSIVE AND NORMOTENSIVE RATS

Objective: Systemic hypertension has a profound impact on the renal vascular physiology and functionality. Our goal was to identify the biological pathways and macromolecules of the renal arteriolar wall, which are involved with the development of hypertrension. Design and method: Tissue derived exclusively from renal vessels of 4 Spontaneously Hypertensive Rat (SHR) and 4 normotensive controls (Wistar Kyoto,WKY) at 20 weeks using Laser Capture Microdissection on 14 micrometer cryosections was used. High sensitivity proteomic analysis was performed in the microdissected homogenized material in order to detect early molecular alterations associated with hypertension of the renal vessels before the onset of vascular damage. Results: Proteomic analysis revealed 688 proteins; 550 proteins were found in both groups, of which 58 proteins were differentially expressed (15 proteins were up-regulated and 43 proteins were down-regulated in SHR). 71 proteins were found exclusively in control WKY rats and 67 exclusively in SHR rats. Pathway enrichment analysis revealed 114 and 111 pathways in WKY and SHR, respectively and 106 common pathways in both groups. Many of the interesting differentially expressed proteins identified in our study are relevant to vascular tone regulation. Thus proteins involved with NO and vasodilation and affecting eNOS include Xaa-Pro aminopeptidase 1 (XPP1), N (G) N (G)-dimethylarginine dimethylaminohydrolase 1 (DDAH1), Dehydropteridine reductase (DHPR), whereas proteins involved with blood pressure regulation by the renin-angiotensin system include Glutamyl aminopeptidase / Aminopeptidase A (AMPE) and Aminopeptidase N (AMPN). Moreover, pathway enrichment analysis revealed that the eNOS activation pathway is disregulated only in the hypertensive SHR animals. Conclusions: Our study demonstrates that hypertension causes early proteomic changes in the renal vessels of SHR compared to WKY. These changes are relevant to vascular tone regulation and consequently may be involved in the development of vascular damage and hypertensive nephrosclerosis. Further studies are required to explore whether these pathways and molecules are involved with hypertensive nephrosclerosis and to identify components that could be considered new therapeutic targets

ITRAQ-based Proteomic Analysis of APPSw,Ind Mice Provides Insights into the Early Changes in Alzheimer's Disease.

Background: Several proteins have been identified as potential diagnostic biomarkers in imaging, genetic, or proteomic studies in Alzheimer disease (AD) patients and mouse models. However, biomarkers for presymptom diagnosis of AD are still under investigation, as are the presymptom molecular changes in AD pathogenesis.Objective: In this study, we aim to analyzed the early proteomic changes in APPSw,Ind mice and to conduct further functional studies on interesting proteins.Methods: We used the isobaric tags for relative and absolute quantitation (iTRAQ) approach combined with mass spectrometry to examine the early proteomic changes in hippocampi of APPSw,Ind mice. Quantitative reverse transcription polymerase chain reaction (RT-PCR) and immuno-blotting were performed for further validation. Finally, the functions of interesting proteins β-spectrin and Rab3a in APP trafficking and processing were tested by shRNA knockdown, in N2A cells stably expressing β-amyloid precursor protein (APP).Results: The iTRAQ and RT-PCR results revealed the detailed molecular changes in oxidative stress, myelination, astrocyte activation, mTOR signaling and Rab3-dependent APP trafficking in the early stage of AD progression. Knock down of β-spectrin and Rab3a finally led to increased APP fragment production, indicating key roles of β-spectrin and Rab3a in regulating APP processing.Conclusion: Our study provides the first insights into the proteomic changes that occur in the hippocampus in the early stages of the AD mouse model. In addition to improving the understanding of molecular alterations and functional cascades involved in early AD pathogenesis, our findings raise the possibility of developing potential biomarkers and therapeutic targets for early AD.

Early proteomic changes in pear (Pyrus communis L.) calli induced by co-culture on microcallus suspension of incompatible quince (Cydonia oblonga Mill.)

The pear (Pyrus communis L.)/quince (Cydonia oblonga Mill.) graft incompatibility is associated to biochemical and morphological alterations impairing the establishment of a functional union between the two partners, in which the callus at graft interface plays pivotal roles. This work was aimed to study the early effects evoked by factor(s) released by quince cells on the metabolism of pear callus. Using two-dimensional difference gel electrophoresis, the study analysed the changes between the proteome of pear callus co-cultured on suspensions of pear microcalli (homologous combination) or of incompatible quince microcalli (heterologous combination) by an in vitro system. This approach revealed changes in the abundance of proteins involved in several metabolic pathways and cell processes. In pear callus in heterologous combination, the less abundance of α-amylase and enolase and the increase of enzymes involved in amino acid and pyrimidine recycling indicate an alteration of the carbohydrate usage and a concomitant upsurge of salvage pathways. Moreover, the higher accumulation of a polyphenol oxidase suggests a participation of the flavonoid metabolism in the protection from the stress evoked by the proximity of quince cells. At the same time, this stress condition resulted associated with a reduction of proteins involved in the cell translation machinery and vesicle traffic, supporting that in heterologous combination the pear callus undergoes a physiological status less favourable for proliferation and differentiation. Overall, the work provides new information about the pear/quince incompatibility, highlighting as it involves a network of different metabolic responses.

Proteomic analysis of anatoxin-a acute toxicity in zebrafish reveals gender specific responses and additional mechanisms of cell stress

Anatoxin-a is a potent neurotoxin produced by several genera of cyanobacteria. Deaths of wild and domestic animals due to anatoxin-a exposure have been reported following a toxic response that is driven by the inhibition of the acetylcholine receptors at neuromuscular junctions. The consequent neuron depolarization results in an overstimulation of the muscle cells. In order to unravel further molecular events implicated in the toxicity of anatoxin-a, a proteomic investigation was conducted. Applying two-dimensional gel electrophoresis (2DE) and MALDI-TOF mass spectrometry, we report early proteome changes in brain and muscle of zebrafish (Danio rerio) caused by acute exposure to anatoxin-a. In this regard, the test group of male and female zebrafish received an intraperitoneal (i.p.) injection of an anatoxin-a dose of 0.8µgg−1 of fish body weight (bw) in phosphate buffered saline solution (PBS), while the control received an i.p. injection of PBS only. Five minutes after i.p. injection, brain and muscle tissues were collected, processed and analyzed with 2DE. Qualitative and quantitative analyzes of protein abundance allowed the detection of differences in the proteome of control and exposed fish groups, and between male and female fish (gender specific responses). The altered proteins play functions in carbohydrate metabolism and energy production, ATP synthesis, cell structure maintenance, cellular transport, protein folding, stress response, detoxification and protease inhibition. These changes provide additional insights relative to the toxicity of anatoxin-a in fish. Taking into account the short time of response considered (5min of response to the toxin), the changes in the proteome observed in this work are more likely to derive from fast occurring reactions in the cells. These could occur by protein activity regulation through degradation (proteolysis) and/or post-translational modifications, than from a differential regulation of gene expression, which may require more time for proteins to be synthesized and to produce changes at the proteomic level.

Dynamic changes in rat mesenteric lymph proteins following trauma using label-free mass spectrometry.

Early events triggered by posttrauma/hemorrhagic shock currently represent a leading cause of morbidity and mortality in these patients. The causative agents of these events have been associated with increased neutrophil priming secondary to shock-dependent alterations of mesenteric lymph. Previous studies have suggested that unknown soluble components of the postshock mesenteric lymph are main drivers of these events. In the present study, we applied a label-free proteomics approach to further delve into the early proteome changes of the mesenteric lymph in response to hemorrhagic shock. Time-course analyses were performed by sampling the lymph every 30 min after shock up until 3 h (the time window within which a climax in neutrophil priming was observed). There are novel, transient early post-hemorrhagic shock alterations to the proteome and previously undocumented postshock protein alterations. These results underlie the triggering of coagulation and proinflammatory responses secondary to trauma/hemorrhagic shock, metabolic deregulation and apoptosis, and alterations to proteases/antiproteases homeostasis, which are suggestive of the potential implication of extracellular matrix proteases in priming neutrophil activation. Finally, there is a likely correlation between early postshock mesenteric lymph-mediated neutrophil priming and proteomics changes, above all protease/antiproteases impaired homeostasis (especially of serine proteases and metalloproteases).

Investigation of early proteomic changes in liver affected by non-alcoholic steatohepatitis

The increasing number of obesity in western society is a major cause for higher incidence of metabolic aberrations as insulin resistance and non-alcoholic fatty liver disease (NAFLD). Non-alcoholic steatohepatitis (NASH) is the most severe form of NAFLD, characterized by liver steatosis and inflammation leading to a significant deterioration in liver function, possibly culminating in liver cirrhosis. However, the exact mechanism of NASH development is still unclear.An established mouse model system for NASH is used to assess the early changes in hepatic protein regulation. Wild-type mice are fed with a high-fat diet facilitating the development of adipositas and NASH. Proteomic changes in different compartments of murine liver cells are investigated in a time course. The analyses of protein-related changes are accomplished by means of two-dimensional differential gel electrophoresis (DIGE), followed by protein identification using liquid chromatography-mass spectrometry. In a complementary approach, changes in protein abundances are quantified by a shotgun approach using stable-istotope labelling. Furthermore, results will be confirmed using functional assays. The grade of steatosis was determined by histological examination. During the course of time, a microvesicular steatosis with mild inflammatory infiltration was confirmed. The degree of inflammation and fibrosis was assessed by quantitative real-time-PCR of relevant marker genes. The liver proteome was examined for differentially regulated proteins by DIGE. An upregulation of various proteins involved in lipid metabolism as well as primary energy metabolism could be shown. Also, various proteins participating in one carbon metabolism were shown to be regulated in early disease progression. For several proteins, a graduated regulation over the experimental course was observed.

Early Changes in Protein Expression Detected by Mass Spectrometry Predict Tumor Response to Molecular Therapeutics

Biomarkers that predict therapeutic response are essential for the development of anticancer therapies. We have used matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) to directly analyze protein profiles in mouse mammary tumor virus/HER2 transgenic mouse frozen tumor sections after treatment with the erbB receptor inhibitors OSI-774 and Herceptin. Inhibition of tumor cell proliferation and induction of apoptosis and tumor reduction were predicted by a >80% reduction in thymosin beta4 and ubiquitin levels that were detectable after 16 hours of a single drug dose before any evidence of in situ cellular activity. These effects were time- and dose-dependent, and their spatial distribution in the tumor correlated with that of the small-molecule inhibitor OSI-774. In addition, they predicted for therapeutic synergy of OSI-774 and Herceptin as well as for drug resistance. These results suggest that drug-induced early proteomic changes as measured by MALDI-MS can be used to predict the therapeutic response to established and novel therapies.