Oxidative Stress In Cancer Aids And Neurodegenerative Diseases Pdf
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- Low Concentrations of Acid-Soluble Thiol (Cysteine) in the Blood Plasma of HIV-1-Infected Patients
- Oxidative Stress and Disease
- Oxidative Stress and Neurodegenerative Disease
- Oxidative Stress in Infection and Consequent Disease
Typically in aerobic metabolism, organic compounds such as nucleic acids, proteins and lipids can undergo structural damage by oxidative reactions. Various diseases such as cancer, diabetes, cardiovascular diseases and neurodegenerative clearly exemplify the chronic oxidative stress. Therefore, it is important to consider that at low and moderate ROS levels, it can, for example, act as signaling molecules that support cell proliferation and differentiation and activate survival pathways in response to stress.
Low Concentrations of Acid-Soluble Thiol (Cysteine) in the Blood Plasma of HIV-1-Infected Patients
Oxidatively damaged proteins accumulate with age in almost all cell types and tissues. The activity of chaperone-mediated autophagy CMA , a selective pathway for the degradation of cytosolic proteins in lysosomes, decreases with age. We have analyzed the possible participation of CMA in the removal of oxidized proteins in rat liver and cultured mouse fibroblasts.
Added to the fact that CMA substrates, when oxidized, are more efficiently internalized into lysosomes, we have found a constitutive activation of CMA during oxidative stress. Oxidation-induced activation of CMA correlates with higher levels of several components of the lysosomal translocation complex, but in particular of the lumenal chaperone, required for substrate uptake, and of the lysosomal membrane protein lamp type 2a, previously identified as a receptor for this pathway.
In contrast with the well characterized mechanism of CMA activation during nutritional stress, which does not require de novo synthesis of the receptor, oxidation-induced activation of CMA is attained through transcriptional up-regulation of lamp2a. We conclude that CMA is activated during oxidative stress and that the higher activity of this pathway under these conditions, along with the higher susceptibility of the oxidized proteins to be taken up by lysosomes, both contribute to the efficient removal of oxidized proteins.
Accumulation of oxidized protein is a common feature of aged cells Dunlop et al. Many physiological and pathological processes lead to the generation of free radicals and consequent damage of intracellular components, including proteins. In most of these oxidative events, damaged proteins are removed from the cell through degradation by proteases Dunlop et al. The activity of different intracellular proteolytic systems decreases with age Terman, ; Cuervo and Dice, a ; Friguet et al.
The susceptibility of oxidized proteins to proteases changes with the duration and degree of oxidative damage Dunlop et al. Thus, mild oxidation induces partial protein unfolding and facilitates proteolytic cleavage Grune et al.
However, persistent or extensive oxidative damage usually promotes protein aggregation, due to the exposure of patches of hydrophobic amino acids. Once a protein aggregates, it becomes less susceptible to proteolytic cleavage Hoff et al.
Kinetics of degradation of oxidized proteins in vitro have been analyzed using different types of proteases Merker and Grune, ; Dunlop et al. One of the most extensively analyzed protease in this respect has been the proteasome. Although most of the studies linking the proteasome to the degradation of oxidized proteins have been carried out in vitro Rivett, ; Davies, , recent evidence supports the participation of the proteasome in the removal of oxidized proteins in vivo also Hosler et al.
Thus, treatment of culture cells with proteasome inhibitors or antisense oligonucleotides against essential proteasome subunits results in accumulation of oxidized proteins inside cells and diminished rates of cell survival during oxidative stress. The degradation of most oxidized proteins by the proteasome is ATP and ubiquitin independent Shringarpure et al. The lysosomal system, the other major proteolytic system in cells, has not been often considered as a possible candidate for the removal of oxidized proteins because of its lack of selectivity.
In mammalian cells three different main mechanisms contribute to the degradation of intracellular components inside lysosomes autophagy Dice, ; Reggiori and Klionsky, ; Wang and Klionsky, ; Cuervo, a , b. Two of those mechanisms, macroautophagy and microautophagy, result in the degradation of complete regions of the cytosol, including organelles, in the lysosomal lumen.
Although degradation of organelles by these autophagic pathways can be selective i. In contrast, the main characteristic of a third form of autophagy, chaperone-mediated autophagy CMA , is its selectivity regarding the substrates cytosolic proteins degraded through this pathway Cuervo and Dice, b ; Dice, ; Dice et al. CMA is a generalized form of autophagy present in most cell types and tissues Massey et al. The interaction with this chaperone, modulated by the hsc70 cochaperones Agarraberes and Dice, , targets the substrate to the lysosomal membrane, where it interacts with the lysosomal membrane protein lamp type 2a Cuervo and Dice, Substrates need to be unfolded before translocation into the lysosomal lumen, and several cytosolic chaperones associated to the lysosomal membrane have been proposed to assist in the unfolding Agarraberes and Dice, Translocation of the substrate requires the presence of a variant of hsc70, lyshsc70, in the lysosomal lumen Agarraberes et al.
Although some basal level of CMA activity is probably present in most cells, nutritional stress has been shown to maximally activate this pathway Wing et al. Activation during nutrient deprivation is associated with higher levels of lys-hsc70 in the lysosomal lumen and of lamp2a at the lysosomal membrane Cuervo et al.
Because the interaction of substrate proteins with lamp2a is a limiting step for this pathway, changes in levels of lamp2a at the lysosomal membrane modulate CMA activity Cuervo and Dice, b , c.
Interestingly, all the conditions known to activate CMA, up-regulation of the lysosomal levels of lamp2a does not require de novo synthesis of the protein, but instead it is attained through down-regulation of its degradation and by relocation of a fraction of the protein from the lysosomal lumen into the membrane Cuervo and Dice, b.
In addition to starvation, activation of CMA has also been observed in rat liver and kidney after exposure to gasoline derivatives Cuervo et al.
CMA activity is reduced during renal tubular cell growth Franch et al. The decrease in CMA activity in old cells, known to accumulate oxidized proteins Cuervo and Dice, a , combined with the fact that activation of CMA during toxic exposure results in the selective degradation of a protein altered by the toxic compound Cuervo et al. We show in this work that oxidation of CMA substrates facilitates their translocation into lysosomes for degradation via CMA and also that CMA itself is activated during oxidative stress.
Unexpectedly, the oxidative stress-mediated activation of CMA is attained through a novel mechanism, different from the previous well-characterized activation of CMA in response to nutritional stress. Male Wistar rats — g were fed ad libitum or starved for 20 or 48 h before sacrifice. An age-controlled rat strain Fisher was used for the study of age-related changes and 3-, , and mo-old rats were compared.
Mild oxidative stress was induced in rats with two single i. Lysosomal isolation was carried out 24 h after the last injection. To deprive cells from serum, plates were extensively washed with Hanks' balanced salt solution Invitrogen, Carlsbad, CA , and fresh medium without serum was added.
Assays were carried out 12—24 h after removing the oxidizing compound. Sources of chemicals and antibodies were as described previously Cuervo and Dice, ; Cuervo et al. The antibodies against the cytosolic tail of rat and mouse lamp2a and lamp2c were prepared in our laboratory Cuervo and Dice, ; Zhang, Bandhyopadhyay, Kiffin, Massey, and Cuervo, unpublished data.
Carbonyl groups in oxidized proteins were detected using the OxyBlot oxidized protein detection kit from Chemicon International Temecula, CA. Oxidized radiolabeled cytosolic proteins were prepared by the same procedure from cells treated with different prooxidants for 24 h before isolation. Rat liver lysosomes were isolated from a light mitochondrial-lysosomal fraction in a discontinuous metrizamide density gradient Wattiaux et al.
Lysosomal fractions with different activities for CMA were separated as described previously Cuervo et al. Lysosomes from cultured cells were isolated as described previously Storrie and Madden, Lysosomal matrices and membranes were isolated after hypotonic shock Ohsumi et al.
In another group of experiments, to overcome any possible changes in the proteolytic susceptibility of the oxidized proteins to proteinase K, substrates were incubated with chymostatin-treated or untreated lysosomes, and uptake was calculated as the difference between the amount of substrate associated to lysosomes chymostatin-treated lysosomes and the amount of substrate bound to their membrane untreated lysosomes Salvador et al.
Radioactivity in the flow through and in the filter was converted to disintegrations per minute in a WinSpectral liquid scintillation analyzer PerkinElmer Life and Analytical Sciences, Boston, MA by correcting for quenching using an external standard.
Proteolysis was expressed as the percentage of the initial acid-insoluble radioactivity protein transformed into acid-soluble radioactivity amino acids and small peptides at the end of the incubation.
Rates of degradation of lamp2a in the isolated membranes were followed by immunoblot with a specific antibody against the cytosolic tail of lamp2a as described previously Cuervo and Dice, b.
Metal-catalyzed oxidation of rabbit muscle d -glyceraldehydephosphate dehydrogenase EC 1. The efficiency of oxidation was monitored by measuring the enzymatic activity of GAPDH after being exposed to the oxidant mixture for different periods of time. Figure 2. Oxidation of CMA substrates facilitates their uptake by lysosomes.
B Unmodified GAPDH Ctr or GAPDH exposed to the oxidant mixture for the indicated periods of time was incubated for 30 min under standard conditions with intact chymostatin-treated liver lysosomes isolated from h starved rats. Where indicated, samples were treated with proteinase K to remove GAPDH bound to the cytosolic side of the lysosomal membrane.
D Mouse fibroblasts were metabolically labeled with [ 3 H]leucine for 48 h. At the end of the incubation proteolysis was calculated as the amount of acid precipitable radioactivity protein transformed in acid soluble amino acids and small peptides. Immunofluorescence studies of cultured cells were performed following conventional procedures Cuervo and Dice, c.
Mounting medium contained 4,6-diamidinophenylindole staining to highlight the cellular nucleus. Images were acquired with an Axiovert fluorescence microscope Carl Zeiss, Thornwood, NY and subjected to deconvolution with the manufacturer's software. All digital microscopic images were prepared using Adobe Photoshop 6. For both genes, the presence of a single amplified product was verified by agarose gel electrophoresis, and by analysis of the melting curves of the reverse transcription-PCR reaction.
The expression levels of lamp2a in different samples were normalized with respect of those of actin in the same samples. Differences between samples were calculated based in their differences of the cycle numbers to reach a certain fluorescence intensity level. Because the size of the amplified fragments was very similar we did not need to correct for fragment length. PCR products were subjected to electrophoresis in 1.
The densitometric intensity of the lamp2a products was normalized with respect to the intensity of the actin products for the same sample. Protein concentration was determined by the Lowry method Lowry et al. Lysosomal enzymatic activities were measured as reported previously Storrie and Madden, Oxidized proteins were visualized after derivatization with DNPH by immunoblot with an antibody against the DNP moiety following the manufacturer's recommendation.
Student's t test was used for statistical analyses. To test the contribution that the lysosomal system, and in particular CMA, might play in the removal of oxidized cytosolic proteins, we first analyzed the presence of those proteins inside lysosomes after oxidative stress.
To verify the oxidative effect of paraquat in rat liver we compared the content of carbonyl-containing proteins in the cytosol of untreated and paraquat-treated rats Figure 1A , lanes 1 and 2. Although some oxidized proteins can be observed in the cytosol of untreated rats, we found a significant increase in the content of these modified proteins in the cytosol of rats injected with a sublethal dose of paraquat for two consecutive days.
Lysosomes active for CMA can be isolated from rat liver based on their high content of the lumenal chaperone lys-hsc70 required for substrate uptake Cuervo et al. When we isolated this particular lysosomal population from treated and untreated rats and separated their membranes from their lumenal content, we found carbonyl-containing proteins in both the membrane and lumenal fraction of the lysosomes from paraquat-treated rats Figure 1A , compare lanes lanes 3 and 5 to lanes 4 and 6.
Washing the lysosomal membranes with 1 M NaCl released most of the oxidized proteins Figure 1A , lanes 7 and 8 , suggesting that they were likely cytosolic proteins associated to the membrane, rather than the result of oxidation of integral lysosomal membrane proteins. These results support that the oxidized proteins detected inside the lysosomes were most likely nonlysosomal proteins delivered there to be degraded.
Interestingly, despite of their cytosolic origin, the electrophoretic pattern of the oxidized proteins associated with the lysosomal fraction differs from the ones in the cytosol. Because these differences are also observed in the membrane associated proteins, we do not think that they are due to distinct susceptibility of some of the oxidized proteins to proteases; instead we consider it an indication that a subset of oxidized proteins is selectively taken up by lysosomes.
Figure 1. Oxidized proteins in lysosomes active for CMA. To show the protein pattern in all lanes, exposure of lanes 1 and 2 was 5 times shorter than for the other lanes. Lanes 7 and 8 are duplicate samples of lanes 3 and 4, in which membranes were washed with 1 M NaCl before derivatization.
No bands were detected in the same samples when derivatization was omitted our unpublished data. Content of oxidized proteins in these fractions was analyzed as in A. Exposure of lanes 1—3 was 3 times shorter than for lanes 4—6. Lanes 7 and 8 show the same sample than lane 5 derivatized or not with DPNH and immunobloted as the others. The film is overexposed 8 times lane 5 exposure to show the low content of protein bands nonspecifically recognized by the antibody.
As reported previously, even in the absence of treatment with a prooxidant agent, oxidized proteins can be detected in normal liver and their content increases with age Figure 1B. When we analyzed the association of oxidized proteins to lysosomes from livers of 3- and 9-mo-old rats, we found an increase in the content of oxidized proteins in the lysosomal lumen with age, similar to the one observed in the cytosol Figure 1B , compare lanes 1 and 2 and 4 and 5.
In contrast, in the oldest animals 22 mo old , for which we have previously described a severe decrease in CMA activity Cuervo and Dice, a , despite the higher content of oxidized proteins in the cytosolic fraction, in proportion, the amount of oxidized proteins detected in lysosomes was lower than in the 9-mo-old animals Figure 1B , compare lanes 5 and 6.
Oxidative Stress and Disease
Oxidatively damaged proteins accumulate with age in almost all cell types and tissues. The activity of chaperone-mediated autophagy CMA , a selective pathway for the degradation of cytosolic proteins in lysosomes, decreases with age. We have analyzed the possible participation of CMA in the removal of oxidized proteins in rat liver and cultured mouse fibroblasts. Added to the fact that CMA substrates, when oxidized, are more efficiently internalized into lysosomes, we have found a constitutive activation of CMA during oxidative stress. Oxidation-induced activation of CMA correlates with higher levels of several components of the lysosomal translocation complex, but in particular of the lumenal chaperone, required for substrate uptake, and of the lysosomal membrane protein lamp type 2a, previously identified as a receptor for this pathway.
Oxidative Stress and Neurodegenerative Disease
Oxidative stress OS has the ability to damage different molecules and cellular structures, altering the correct function of organs and systems. OS accumulates in the body by endogenous and exogenous mechanisms. Increasing evidence points to the involvement of OS in the physiopathology of various chronic diseases that require prolonged periods of pharmacological treatment.
Materials and Methods: A total of 28 AD, 42 PD patients and 42 healthy controls aged yrs were recruited for the study. This indicates that reduced SOD plays a prominent role in the increase of OS in neuronal degeneration. This article has been cited by 1 Neuroprotective effect of Reinwardtia indica against scopolamine induced memory-impairment in rat by attenuating oxidative stress Prabhat Upadhyay,Rashmi Shukla,Kavindra Nath Tiwari,G. Tantisira,Ratchanee Rodsiri Phytomedicine. Carvalho,Michael Maes,Adam J.
Oxidative Stress in Infection and Consequent Disease
If the address matches an existing account you will receive an email with instructions to reset your password. If the address matches an existing account you will receive an email with instructions to retrieve your username. Ataxia telangiectasia AT is a pleiotropic genetic disorder characterized by progressive neurodegeneration, especially of cerebellar Purkinje cells, immunodeficiency, increased incidence of cancer, and premature aging.
Alexander V. Ivanov, Birke Bartosch, Maria G. Viral, bacterial, and parasitic infections comprise a vast group of etiological agents that cause acute or chronic diseases. According to WHO, they represent one of the major causes of human morbidity and mortality.
Free radicals are common outcome of normal aerobic cellular metabolism. In-built antioxidant system of body plays its decisive role in prevention of any loss due to free radicals. However, imbalanced defense mechanism of antioxidants, overproduction or incorporation of free radicals from environment to living system leads to serious penalty leading to neuro-degeneration. Neural cells suffer functional or sensory loss in neurodegenerative diseases. Apart from several other environmental or genetic factors, oxidative stress OS leading to free radical attack on neural cells contributes calamitous role to neuro-degeneration.
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