The focus of this review is on the proteomic approaches applied to the study of the qualitative/quantitative changes in mitochondrial proteins that are related to impaired mitochondrial function and consequently different types of pathologies. Proteomic techniques developed in recent years have created a powerful tool for the characterization of both static and dynamic proteomes. They can detect protein-protein interactions and a broad repertoire of post-translation modifications that play pivotal roles in mitochondrial regulation, maintenance and proper function. Based on accumulated proteomic data, conclusions can be derived on how to proceed in disease prevention and treatment. In addition, this article will present an overview of the recently published proteomic papers that deal with the regulatory roles of post-translational modifications of mitochondrial proteins and specifically with cardiovascular diseases connected to mitochondrial dysfunction.
Cardiovascular disease is the main cause of death worldwide, making it crucial to search for new therapies to mitigate major adverse cardiac events (MACEs) after a cardiac ischemic episode. Drugs in the class of the glucagon-like peptide-1 receptor agonists (GLP1Ra) have demonstrated benefits for heart function and reduced the incidence of MACE in patients with diabetes. Previously, we demonstrated that a short-acting GLP1Ra known as DMB (2-quinoxalinamine, 6,7-dichloro-N-[1,1-dimethylethyl]-3-[methylsulfonyl]-,6,7-dichloro-2-methylsulfonyl-3-N-tert-butylaminoquinoxaline or compound 2, Sigma) also mitigates adverse postinfarction left ventricular remodeling and cardiac dysfunction in lean mice through activation of parkin-mediated mitophagy following infarction. Here, we combined proteomics with in silico analysis to characterize the range of effects of DMB in vivo throughout the course of early postinfarction remodeling. We demonstrate that the mitochondrion is a key target of DMB and mitochondrial respiration, oxidative phosphorylation and metabolic processes such as glycolysis and fatty acid beta-oxidation are the main biological processes being regulated by this compound in the heart. Moreover, the overexpression of proteins with hub properties identified by protein-protein interaction networks, such as Atp2a2, may also be important to the mechanism of action of DMB. Data are available via ProteomeXchange with identifier PXD027867.
- MeSH
- chinoxaliny aplikace a dávkování farmakologie MeSH
- glykolýza MeSH
- mapy interakcí proteinů MeSH
- modely nemocí na zvířatech MeSH
- myši MeSH
- oxidativní fosforylace MeSH
- proteomika metody MeSH
- receptor pro glukagonu podobný peptid 1 agonisté MeSH
- remodelace komor účinky léků MeSH
- sarkoplazmatická Ca2+-ATPáza metabolismus MeSH
- srdeční komory metabolismus MeSH
- srdeční mitochondrie metabolismus MeSH
- výpočetní biologie MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Continuous flow electrophoretic separation with continuous sample loading provides the advantage of processing volumes of any sizes, as well as the benefit of a real-time monitoring and optimization of the separation process. In addition, the spatial separation of the sample enables collecting multiple separated components simultaneously and in a continuous manner. The separation is usually performed in mild buffers without organic solvents and detergents (sample biological activity is retained) and it is carried out without usage of a solid support in the separation space preventing the interaction of the sample with it (high sample recovery). The method is used for the separation of proteins/peptides in proteomic applications, and its great applicability is to the separation of the cells, cellular organelles, vesicles, membrane fragments, and DNA. This review focuses on the electrophoretic separation performed in a continuous flow and it describes various electrophoretic modes and instrumental setups. Recent developments in methodology and instrumentation, the integration with other techniques, and the application to the biological sample analysis are discussed as well.
The effects of ER stress on protein secretion by cardiac myocytes are not well understood. In this study, the ER stressor thapsigargin (TG), which depletes ER calcium, induced death of cultured neonatal rat ventricular myocytes (NRVMs) in high media volume but fostered protection in low media volume. In contrast, another ER stressor, tunicamycin (TM), a protein glycosylation inhibitor, induced NRVM death in all media volumes, suggesting that protective proteins were secreted in response to TG but not TM. Proteomic analyses of TG- and TM-conditioned media showed that the secretion of most proteins was inhibited by TG and TM; however, secretion of several ER-resident proteins, including GRP78 was increased by TG but not TM. Simulated ischemia, which decreases ER/SR calcium also increased secretion of these proteins. Mechanistically, secreted GRP78 was shown to enhance survival of NRVMs by collaborating with a cell-surface protein, CRIPTO, to activate protective AKT signaling and to inhibit death-promoting SMAD2 signaling. Thus, proteins secreted during ER stress mediated by ER calcium depletion can enhance cardiac myocyte viability.
- MeSH
- apoptóza MeSH
- autokrinní signalizace MeSH
- biologické markery MeSH
- epidermální růstový faktor metabolismus MeSH
- kardiomyocyty účinky léků metabolismus MeSH
- krysa rodu rattus MeSH
- kultivované buňky MeSH
- membránové glykoproteiny metabolismus MeSH
- myši MeSH
- náchylnost k nemoci MeSH
- nádorové proteiny metabolismus MeSH
- parakrinní signalizace MeSH
- proteom * MeSH
- proteomika * metody MeSH
- sarkoplazmatické retikulum metabolismus MeSH
- signální transdukce účinky léků MeSH
- stres endoplazmatického retikula * účinky léků MeSH
- thapsigargin farmakologie MeSH
- vápník metabolismus MeSH
- vápníková signalizace účinky léků MeSH
- viabilita buněk MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
Studies in dynamic changes in protein translation require specialized methods. Here we examined changes in newly-synthesized proteins in response to ischemia and reperfusion using the isolated perfused mouse heart coupled with polysome profiling. To further understand the dynamic changes in protein translation, we characterized the mRNAs that were loaded with cytosolic ribosomes (polyribosomes or polysomes) and also recovered mitochondrial polysomes and compared mRNA and protein distribution in the high-efficiency fractions (numerous ribosomes attached to mRNA), low-efficiency (fewer ribosomes attached) which also included mitochondrial polysomes, and the non-translating fractions. miRNAs can also associate with mRNAs that are being translated, thereby reducing the efficiency of translation, we examined the distribution of miRNAs across the fractions. The distribution of mRNAs, miRNAs, and proteins was examined under basal perfused conditions, at the end of 30 min of global no-flow ischemia, and after 30 min of reperfusion. Here we present the methods used to accomplish this analysis-in particular, the approach to optimization of protein extraction from the sucrose gradient, as this has not been described before-and provide some representative results.
- MeSH
- messenger RNA genetika MeSH
- mikro RNA metabolismus MeSH
- myši MeSH
- polyribozomy metabolismus MeSH
- proteomika metody MeSH
- srdce růst a vývoj MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- audiovizuální média MeSH
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
INTRODUCTION: Protein synthesis is the outcome of tightly regulated gene expression which is responsive to a variety of conditions. Efforts are ongoing to monitor individual stages of protein synthesis to ensure maximum efficiency and accuracy. Due to post-transcriptional regulation mechanisms, the correlation between translatome and proteome is higher than between transcriptome and proteome. However, the most accurate approach to assess the key modulators and final protein expression is directly by using proteomics. Areas covered: This review covers various proteomic strategies that were used to better understand post-transcriptional regulation, specifically during and early after translation. The methods that identify both regulatory proteins associated with translational components and newly synthesized proteins are discussed. Expert commentary: Emerging proteomic approaches make it possible to monitor protein dynamics in cells, tissues and whole animals. The ability to detect alteration in protein abundance soon after their synthesis enables earlier recognition of disease causing factors and candidates to prevent/rectify disease phenotype.
- MeSH
- biomedicínský výzkum metody MeSH
- proteomika * MeSH
- proteosyntéza genetika MeSH
- regulace genové exprese genetika MeSH
- ribozomy genetika MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
This study concentrates on development of instrumentation for focusing and separation of analytes in continuous flow. It is based on bidirectional ITP working in wide pH range with separation space of closed void channel of trapezoidal shape and continuous supply of sample. The novel instrumentation is working with electrolyte system formulated previously and on the contrary to devices currently available, it allows preparative separation and concentration of cationic, anionic, and amphoteric analytes simultaneously and in wide pH range. The formation of sharp edges at zone boundaries as well as low conductivity zones are avoided in suggested system and thus, local overheating is eliminated allowing for high current densities at initial stages of focusing. This results in high focusing speed and reduction of analysis time, which is particularly advantageous for separations performed in continuous flow systems. The closed void channel is designed to avoid basic obstacles related to liquid leakage, bubbles formation, contacts with electrodes, channel height and complicated assembling. The performance of designed instrumentation and focusing dynamics were tested by using colored low molecular mass pH indicators for local pH determination, focusing pattern, and completion. In addition, feasibility and separation efficiency were demonstrated by focusing of cytochrome C and myoglobin. The collection of fractions at instrument output allows for subsequent analysis and identification of sample components that are concentrated and conveniently in form of solution for further processing. Since the instrumentation operates with commercially available simple defined buffers and compounds without need of carrier ampholytes background, it is economically favorable.
- MeSH
- barvicí látky chemie izolace a purifikace MeSH
- chemické modely MeSH
- design vybavení MeSH
- izotachoforéza přístrojové vybavení metody MeSH
- koncentrace vodíkových iontů MeSH
- myoglobin chemie izolace a purifikace MeSH
- studie proveditelnosti MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The alteration in proteome composition induced by environmental changes and various pathologies is accompanied by the modifications of proteins by specific cotranslational and PTMs. The type and site stoichiometry of PTMs can affect protein functions, alter cell signaling, and can have acute and chronic effects. The particular interest is drawn to those amino acid residues that can undergo several different PTMs. We hypothesize that these selected amino acid residues are biologically rare and act within the cell as molecular switches. There are, at least, 12 various lysine modifications currently known, several of them have been shown to be competitive and they influence the ability of a particular lysine to be modified by a different PTM. In this review, we discuss the PTMs that occur on lysine, specifically neddylation and sumoylation, and the proteomic approaches that can be applied for the identification and quantification of these PTMs. Of interest are the emerging roles for these modifications in heart disease and what can be inferred from work in other cell types and organs.
- MeSH
- kardiomyocyty chemie metabolismus MeSH
- krysa rodu rattus MeSH
- kultivované buňky MeSH
- lidé MeSH
- lysin * analýza chemie metabolismus MeSH
- molekulární sekvence - údaje MeSH
- myokard patologie MeSH
- myši MeSH
- nemoci srdce metabolismus MeSH
- posttranslační úpravy proteinů * MeSH
- proteom analýza MeSH
- sekvence aminokyselin MeSH
- skot MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- lidé MeSH
- myši MeSH
- skot MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
- Research Support, N.I.H., Extramural MeSH
In this paper, we suggest new electrolyte system for fast preparative electrofocusing in wide pH range. It is based on bidirectional ITP with multiple counterions and spacers created by commercially available defined simple buffers. The migration course of proposed focusing model can be simulated in advance by using separation conditions and electrolyte components that are consequently applied during the experiments. The suggested electrolyte system allows high current densities at the initial stages of focusing without danger of local overheating, which strongly reduces the time needed for analysis completion. The performance of the electrolyte system is demonstrated by the focusing of synthetic colored low molecular weight indicators and proteins in the arrangements with both linear narrow strip and nonwoven fabric sheet with continuous flow.
Efficient separation method is a crucial part of the process in which components of highly complex biological sample are identified and characterized. Based on the principles of recently newly established electrophoretic method called divergent flow IEF (DF IEF), we have tested the DF IEF instrument which is able to operate without the use of background carrier ampholytes. We have verified that during separation and focusing of sample consisting of high numbers of proteins (yeast lysate and wheat flour extract), the pH gradient of preparative DF IEF can be created by autofocusing of the sample components themselves without any addition of carrier ampholytes. In DF IEF, the proteins are separated, desalted and concentrated in one step. The fractions of yeast lysate sample, collected at the DF IEF output and subjected to gel IEF, contained the zones of proteins gradually covering the pI values from 3.7 to 8.5. In our experimental arrangement, the highest number of proteins has been found in fractions with pI values around 5.3 as detected by polyacrylamide gel IEF with CBB staining. During DF IEF, the selected protein bands have been concentrated up to 16.8-fold.
- MeSH
- amfolytové směsi MeSH
- biologické přípravky chemie izolace a purifikace MeSH
- isoelektrická fokusace přístrojové vybavení metody MeSH
- koncentrace vodíkových iontů MeSH
- kvasinky chemie MeSH
- metody pro přípravu analytických vzorků přístrojové vybavení metody MeSH
- proteiny chemie izolace a purifikace MeSH
- pšenice chemie MeSH
- rostlinné extrakty chemie izolace a purifikace MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
