Maintaining mitochondrial homeostasis and energy metabolism is essential for normal cardiac funct... more Maintaining mitochondrial homeostasis and energy metabolism is essential for normal cardiac function. Adenosine-monophosphate activated protein kinase (AMPK) is an energy sensor in the cell that detects and reacts to fluctuations in intracellular AMP: ATP ratio and it is activated by increased AMP levels. Activated AMPK promotes ATP production by inhibiting anabolic consuming pathways and enhancing catabolic pathways. AMPK has been shown to protect the heart under several cardiac conditions including ischemia and starvation. The cardioprotective effects have been attributed partially to its ability to induce autophagy, a cellular degradation pathway that eliminates protein aggregates and damaged organelles through the lysosome. When mitochondria are targeted for degradation through autophagy, it is termed mitophagy. Both autophagy and mitophagy may occur under the same conditions but they do not always go in the same direction, suggesting that they may be regulated by distinct pathways. Despite its ability to positively regulate autophagy, AMPK has not been shown to directly regulate mitophagy. In the present study, we investigated if AMPK is required for mitophagy in the heart using mice that lack AMPK alpha 2 gene (knockout, KO) and express a novel mitophagy reporter known as mito-Rosella, a dual-emission biosensor composed of a mitochondrial targeting sequence and a RFP-GFP fusion protein. Mitophagy events were seen as red puncta on merged confocal microscopic images. These red puncta represent mitochondrial fragments that are being degraded in lysosomes where the pH sensitive GFP is quenched. To assess mitophagy flux, these mice were also treated with lysosomal protease inhibitors pepstatin A and E64-d, which lead to an accumulation of red puncta in mitolysosomes. To our surprise, AMPK alpha 2 KO mouse hearts had markedly increased amount of red puncta than wild type hearts, the number of which was further increased by the lysosomal protease inhibitors, suggesting an enhanced mitophagy flux in the absence of AMPK alpha 2 gene. Consistently, western blot analysis showed significantly increased autophagy marker protein LC3-II in AMPK alpha 2 KO mouse hearts. These findings were confirmed in H9c2 cardiac myoblast cells treated with siRNAs targeting both AMPK alpha 1 and alpha 2 genes. In addition, the protein expression levels of FUN14 domain containing 1 (FUNDC1), a positive regulator of mitophagy, were up-regulated in the AMPK alpha 2 KO hearts, which may contribute to the enhanced mitophagy. Collectively, these results suggest that AMPK is a negative regulator of mitophagy, contrary to the widely held hypothesis that AMPK signalling is necessary for mitophagy. Future studies are warranted to investigate if AMPK over expression can inhibit mitophagy either at basal level or in response to various cardiac stresses.
Small molecules are optimally lipophilic in nature, which allows for transit through the circulat... more Small molecules are optimally lipophilic in nature, which allows for transit through the circulation by binding to plasma proteins and passage through cellular membranes to gain access to intracellular targets. The molecular composition and architecture of mitochondria is also responsible for attracting certain small molecules that results in accumulation and impact on mitochondrial function. Binding to plasma protein provides a reservoir of the drug capable of providing a longer‐lasting reservoir of the drug compared with those more freely soluble in the bloodstream and may eventually accumulate in target tissues to a greater extent than the latter more hydrophilic drugs. Molecules transported by albumin are released in regions of low drug concentration. This mechanism enables drugs to be delivered to target tissues and enter into cells either by transport through specific plasma membrane transporters or via diffusion through the plasma membrane.
Small-molecule inhibitors of caspases can be modified with moieties such as biotin or fluorescent... more Small-molecule inhibitors of caspases can be modified with moieties such as biotin or fluorescent molecules. After the inhibitor molecule has bound to an active caspase, the caspase itself becomes labeled and can be isolated using affinity purification. This protocol describes the use of the biotinylated pan-caspase inhibitor VAD-FMK and streptavidin beads to isolate active caspases. These caspases are then separated by gel electrophoresis and identified with caspase-specific antibodies using western blotting techniques. Other caspase inhibitors bound with biotin or other labels can be substituted in this assay; labeled inhibitors are available commercially as either pan-caspase or caspase-specific probes.
Monitoring the activity of a caspase, either as an isolated protein or in a complex mixture (e.g.... more Monitoring the activity of a caspase, either as an isolated protein or in a complex mixture (e.g., a cytosolic extract), can be achieved by measuring substrate cleavage. Chromogenic or fluorogenic substrates are available for many caspases. These substrates usually consist of the four-amino-acid motif that is optimal for each caspase and a moiety that, when cleaved, generates either a chromophore or a fluorophore that can be detected using spectrophotometric or fluorimetric means. In this protocol, we describe how to use these substrates to monitor caspase activity in samples containing active caspases (e.g., apoptotic cells). Caspase inhibitors, which contain a moiety that covalently attaches to the active site of the caspase, can be used in these assays. These assays will ascertain whether caspases are involved in a specific process (e.g., whether caspases are activated after an apoptotic stimulus) and are particularly informative if a purified caspase is used. However, the substrates and inhibitors are not specific for a particular caspase in an environment containing multiple caspases. So, if cytosolic or apoptotic cell extracts are used in these assays, additional experiments must be performed to identify exactly which caspases are involved.
Proteomic approaches have been adopted to survey the degradome of caspases during apoptosis. Thes... more Proteomic approaches have been adopted to survey the degradome of caspases during apoptosis. These approaches provide a comprehensive list of substrates and give clues to which pathways are altered during apoptosis by activated caspases. However, substrates identified by large-scale proteomic screening need to be validated as bona fide caspase targets. This ensures that conclusions derived from the screen are based on real substrates and not on artifacts of the proteomic screen. The validation method described in this protocol uses radiolabeled versions of the putative substrates synthesized using in vitro transcription/translation methods. These are incubated with purified caspases to determine whether they are genuine caspase substrates.
Caspases are proteases that are essential components of apoptotic cell death pathways. There are ... more Caspases are proteases that are essential components of apoptotic cell death pathways. There are approximately one dozen apoptotic caspases found in organisms where cells die via apoptosis. These caspases are responsible for initiation or execution of apoptosis through the proteolytic cleavage of specific substrates. These substrates contain specific motifs that are recognized and cleaved by caspases that result in alterations of substrate function that promotes the apoptotic phenotype. Analysis of caspase involvement, much like any other protease, can be followed using peptides corresponding to cleavage motifs of these substrates, which can be used as substrates, inhibitors, or affinity-based probes.Different caspases have different substrates and therefore different motifs are recognized by each different caspase. However, these different caspases have a common amino acid recognition pattern containing an aspartic acid residue at the amino-side of the cleavage site. Therefore, caspase substrates have a certain overlap in the cleavage motif as this aspartic acid is found in almost every one. This means that certain peptide motifs are not exclusively cleaved by one single caspase. This lack of exclusive cleavage has brought the use of these motif-based probes into question and spurred the development of truly caspase-specific motifs. This chapter describes the use of peptide-based probes to measure caspase activity while highlighting the limitations of these reagents.
Maintaining mitochondrial homeostasis and energy metabolism is essential for normal cardiac funct... more Maintaining mitochondrial homeostasis and energy metabolism is essential for normal cardiac function. Adenosine-monophosphate activated protein kinase (AMPK) is an energy sensor in the cell that detects and reacts to fluctuations in intracellular AMP: ATP ratio and it is activated by increased AMP levels. Activated AMPK promotes ATP production by inhibiting anabolic consuming pathways and enhancing catabolic pathways. AMPK has been shown to protect the heart under several cardiac conditions including ischemia and starvation. The cardioprotective effects have been attributed partially to its ability to induce autophagy, a cellular degradation pathway that eliminates protein aggregates and damaged organelles through the lysosome. When mitochondria are targeted for degradation through autophagy, it is termed mitophagy. Both autophagy and mitophagy may occur under the same conditions but they do not always go in the same direction, suggesting that they may be regulated by distinct pathways. Despite its ability to positively regulate autophagy, AMPK has not been shown to directly regulate mitophagy. In the present study, we investigated if AMPK is required for mitophagy in the heart using mice that lack AMPK alpha 2 gene (knockout, KO) and express a novel mitophagy reporter known as mito-Rosella, a dual-emission biosensor composed of a mitochondrial targeting sequence and a RFP-GFP fusion protein. Mitophagy events were seen as red puncta on merged confocal microscopic images. These red puncta represent mitochondrial fragments that are being degraded in lysosomes where the pH sensitive GFP is quenched. To assess mitophagy flux, these mice were also treated with lysosomal protease inhibitors pepstatin A and E64-d, which lead to an accumulation of red puncta in mitolysosomes. To our surprise, AMPK alpha 2 KO mouse hearts had markedly increased amount of red puncta than wild type hearts, the number of which was further increased by the lysosomal protease inhibitors, suggesting an enhanced mitophagy flux in the absence of AMPK alpha 2 gene. Consistently, western blot analysis showed significantly increased autophagy marker protein LC3-II in AMPK alpha 2 KO mouse hearts. These findings were confirmed in H9c2 cardiac myoblast cells treated with siRNAs targeting both AMPK alpha 1 and alpha 2 genes. In addition, the protein expression levels of FUN14 domain containing 1 (FUNDC1), a positive regulator of mitophagy, were up-regulated in the AMPK alpha 2 KO hearts, which may contribute to the enhanced mitophagy. Collectively, these results suggest that AMPK is a negative regulator of mitophagy, contrary to the widely held hypothesis that AMPK signalling is necessary for mitophagy. Future studies are warranted to investigate if AMPK over expression can inhibit mitophagy either at basal level or in response to various cardiac stresses.
Small molecules are optimally lipophilic in nature, which allows for transit through the circulat... more Small molecules are optimally lipophilic in nature, which allows for transit through the circulation by binding to plasma proteins and passage through cellular membranes to gain access to intracellular targets. The molecular composition and architecture of mitochondria is also responsible for attracting certain small molecules that results in accumulation and impact on mitochondrial function. Binding to plasma protein provides a reservoir of the drug capable of providing a longer‐lasting reservoir of the drug compared with those more freely soluble in the bloodstream and may eventually accumulate in target tissues to a greater extent than the latter more hydrophilic drugs. Molecules transported by albumin are released in regions of low drug concentration. This mechanism enables drugs to be delivered to target tissues and enter into cells either by transport through specific plasma membrane transporters or via diffusion through the plasma membrane.
Small-molecule inhibitors of caspases can be modified with moieties such as biotin or fluorescent... more Small-molecule inhibitors of caspases can be modified with moieties such as biotin or fluorescent molecules. After the inhibitor molecule has bound to an active caspase, the caspase itself becomes labeled and can be isolated using affinity purification. This protocol describes the use of the biotinylated pan-caspase inhibitor VAD-FMK and streptavidin beads to isolate active caspases. These caspases are then separated by gel electrophoresis and identified with caspase-specific antibodies using western blotting techniques. Other caspase inhibitors bound with biotin or other labels can be substituted in this assay; labeled inhibitors are available commercially as either pan-caspase or caspase-specific probes.
Monitoring the activity of a caspase, either as an isolated protein or in a complex mixture (e.g.... more Monitoring the activity of a caspase, either as an isolated protein or in a complex mixture (e.g., a cytosolic extract), can be achieved by measuring substrate cleavage. Chromogenic or fluorogenic substrates are available for many caspases. These substrates usually consist of the four-amino-acid motif that is optimal for each caspase and a moiety that, when cleaved, generates either a chromophore or a fluorophore that can be detected using spectrophotometric or fluorimetric means. In this protocol, we describe how to use these substrates to monitor caspase activity in samples containing active caspases (e.g., apoptotic cells). Caspase inhibitors, which contain a moiety that covalently attaches to the active site of the caspase, can be used in these assays. These assays will ascertain whether caspases are involved in a specific process (e.g., whether caspases are activated after an apoptotic stimulus) and are particularly informative if a purified caspase is used. However, the substrates and inhibitors are not specific for a particular caspase in an environment containing multiple caspases. So, if cytosolic or apoptotic cell extracts are used in these assays, additional experiments must be performed to identify exactly which caspases are involved.
Proteomic approaches have been adopted to survey the degradome of caspases during apoptosis. Thes... more Proteomic approaches have been adopted to survey the degradome of caspases during apoptosis. These approaches provide a comprehensive list of substrates and give clues to which pathways are altered during apoptosis by activated caspases. However, substrates identified by large-scale proteomic screening need to be validated as bona fide caspase targets. This ensures that conclusions derived from the screen are based on real substrates and not on artifacts of the proteomic screen. The validation method described in this protocol uses radiolabeled versions of the putative substrates synthesized using in vitro transcription/translation methods. These are incubated with purified caspases to determine whether they are genuine caspase substrates.
Caspases are proteases that are essential components of apoptotic cell death pathways. There are ... more Caspases are proteases that are essential components of apoptotic cell death pathways. There are approximately one dozen apoptotic caspases found in organisms where cells die via apoptosis. These caspases are responsible for initiation or execution of apoptosis through the proteolytic cleavage of specific substrates. These substrates contain specific motifs that are recognized and cleaved by caspases that result in alterations of substrate function that promotes the apoptotic phenotype. Analysis of caspase involvement, much like any other protease, can be followed using peptides corresponding to cleavage motifs of these substrates, which can be used as substrates, inhibitors, or affinity-based probes.Different caspases have different substrates and therefore different motifs are recognized by each different caspase. However, these different caspases have a common amino acid recognition pattern containing an aspartic acid residue at the amino-side of the cleavage site. Therefore, caspase substrates have a certain overlap in the cleavage motif as this aspartic acid is found in almost every one. This means that certain peptide motifs are not exclusively cleaved by one single caspase. This lack of exclusive cleavage has brought the use of these motif-based probes into question and spurred the development of truly caspase-specific motifs. This chapter describes the use of peptide-based probes to measure caspase activity while highlighting the limitations of these reagents.
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