Protein Catabolic Pathways Research Articles

Comparative functional genomics of adaptation to muscular disuse in hibernating mammals.

Hibernation is an energy-saving adaptation that involves a profound suppression of physical activity that can continue for 6-8 months in highly seasonal environments. While immobility and disuse generate muscle loss in most mammalian species, in contrast, hibernating bears and ground squirrels demonstrate limited muscle atrophy over the prolonged periods of physical inactivity during winter, suggesting that hibernating mammals have adaptive mechanisms to prevent disuse muscle atrophy. To identify common transcriptional programmes that underlie molecular mechanisms preventing muscle loss, we conducted a large-scale gene expression screen in hind limb muscles comparing hibernating and summer-active black bears and arctic ground squirrels using custom 9600 probe cDNA microarrays. A molecular pathway analysis showed an elevated proportion of overexpressed genes involved in all stages of protein biosynthesis and ribosome biogenesis in muscle of both species during torpor of hibernation that suggests induction of translation at different hibernation states. The induction of protein biosynthesis probably contributes to attenuation of disuse muscle atrophy through the prolonged periods of immobility of hibernation. The lack of directional changes in genes of protein catabolic pathways does not support the importance of metabolic suppression for preserving muscle mass during winter. Coordinated reduction in multiple genes involved in oxidation-reduction and glucose metabolism detected in both species is consistent with metabolic suppression and lower energy demand in skeletal muscle during inactivity of hibernation.

Protein Catabolism and High Lipid Metabolism Associated with Long-Distance Exercise Are Revealed by Plasma NMR Metabolomics in Endurance Horses

During long distance endurance races, horses undergo high physiological and metabolic stresses. The adaptation processes involve the modulation of the energetic pathways in order to meet the energy demand. The aims were to evaluate the effects of long endurance exercise on the plasma metabolomic profiles and to investigate the relationships with the individual horse performances. The metabolomic profiles of the horses were analyzed using the non–dedicated methodology, NMR spectroscopy and statistical multivariate analysis. The advantage of this method is to investigate several metabolomic pathways at the same time in a single sample. The plasmas were obtained before exercise (BE) and post exercise (PE) from 69 horses competing in three endurance races at national level (130–160 km). Biochemical assays were also performed on the samples taken at PE. The proton NMR spectra were compared using the supervised orthogonal projection on latent structure method according to several factors. Among these factors, the race location was not significant whereas the effect of the race exercise (sample BE vs PE of same horse) was highly discriminating. This result was confirmed by the projection of unpaired samples (only BE or PE sample of different horses). The metabolomic profiles proved that protein, energetic and lipid metabolisms as well as glycoproteins content are highly affected by the long endurance exercise. The BE samples from finisher horses could be discriminated according to the racing speed based on their metabolomic lipid content. The PE samples could be discriminated according to the horse ranking position at the end of the race with lactate as unique correlated metabolite. As a conclusion, the metabolomic profiles of plasmas taken before and after the race provided a better understanding of the high energy demand and protein catabolism pathway that could expose the horses to metabolic disorders.

Latrepirdine improves cognition and arrests progression of neuropathology in an Alzheimer's mouse model

Latrepirdine (Dimebon) is a pro-neurogenic, antihistaminic compound that has yielded mixed results in clinical trials of mild to moderate Alzheimer's disease, with a dramatically positive outcome in a Russian clinical trial that was unconfirmed in a replication trial in the United States. We sought to determine whether latrepirdine (LAT)-stimulated amyloid precursor protein (APP) catabolism is at least partially attributable to regulation of macroautophagy, a highly conserved protein catabolism pathway that is known to be impaired in brains of patients with Alzheimer's disease (AD). We utilized several mammalian cellular models to determine whether LAT regulates mammalian target of rapamycin (mTOR) and Atg5-dependent autophagy. Male TgCRND8 mice were chronically administered LAT prior to behavior analysis in the cued and contextual fear conditioning paradigm, as well as immunohistological and biochemical analysis of AD-related neuropathology. Treatment of cultured mammalian cells with LAT led to enhanced mTOR- and Atg5-dependent autophagy. Latrepirdine treatment of TgCRND8 transgenic mice was associated with improved learning behavior and with a reduction in accumulation of Aβ42 and α-synuclein. We conclude that LAT possesses pro-autophagic properties in addition to the previously reported pro-neurogenic properties, both of which are potentially relevant to the treatment and/or prevention of neurodegenerative diseases. We suggest that elucidation of the molecular mechanism(s) underlying LAT effects on neurogenesis, autophagy and behavior might warranty the further study of LAT as a potentially viable lead compound that might yield more consistent clinical benefit following the optimization of its pro-neurogenic, pro-autophagic and/or pro-cognitive activities.

Role of Nitrite in Tumor Growth, Symbiogenetic Evolution of Cancer Cells, and China’s Successes in the War against Cancer

Statistics and experiments indicate a correlation between cancer mortality and nitrite in drinking water. Nitrite is a reductant that can deprive a cell of oxygen; it is also an oxidant that can be a substrate in anaerobic ammonium oxidation, the metabolic mode of the anammox bacteria. Eukaryote cells evolved through a fusion of anaerobic and aerobic bacteria. We postulate that an anammox bacterium sought refuge in a fusion with a membrane-bound cluster of aerobic bacteria. While the latter evolved into mitochondria organelles, the former became the nucleus of a prokaryote cell. Eventually, oxidative phosphorilation is the characteristic metabolic pathway of normal eukaryote cells, and we postulate that anammox is the protein-catabolism pathway for cancer cells. The metabolism consumes nitrite and explains thus the link between nitrite and cancer.

Formation of distinct inclusion bodies by inhibition of ubiquitin–proteasome and autophagy–lysosome pathways

Accumulation of misfolded proteins is caused by the impairment of protein quality control systems, such as ubiquitin–proteasome pathway (UPP) and autophagy–lysosome pathway (ALP). In this study, the formation of inclusion bodies was examined after the blockade of UPP and/or ALP in A549 cells. UPP inhibition induced a single and large inclusion body localized in microtubule-organizing center. Interestingly, however, ALP inhibition generated dispersed small inclusion bodies in the cytoplasm. Tuberous sclerosis complex 2 was selectively accumulated in the inclusion bodies of UPP-inhibited cells, but not those of ALP-inhibited cells. Blockade of transcription and translation entirely inhibited the formation of inclusion body induced by UPP inhibition, but partially by ALP inhibition. Moreover, the simultaneous inhibition of two protein catabolic pathways independently developed two distinct inclusion bodies within a single cell. These findings clearly demonstrated that dysfunction of each catabolic pathway induced formation and accumulation of unique inclusion bodies on the basis of morphology, localization and formation process in A549 cells.

Abstract 3247: Establishment of a castration-resistant prostate cancer model, JDCaP-hr: Implications for prostate cancer recurrence

Abstract There are few in vivo models of androgen-dependent prostate cancer (ADPC), and limited availability of the models makes it difficult to evaluate anticancer agents for early stage prostate cancer and to investigate the recurrence of ADPC. We previously reported JDCaP xenograft model, an ADPC model with wild type androgen receptor (AR) and TMPRSS2-ERG gene fusion, derived from skin metastatic lesions of a prostate cancer patient. JDCaP xenografts completely regressed after surgical or medical castration; however, some of them relapsed several months after castration. The relapsed JDCaP subline, designated JDCaP-hr (hormone refractory), was established by serial passage in castrated nude mice. The AR antagonist bicalutamide did not show any effect on tumor growth of JDCaP-hr, while testosterone treatment induced tumor regression of JDCaP-hr, indicating that ligand binding to AR is not necessary, rather cytotoxic, for the growth of JDCaP-hr. Direct sequencing analysis of full length AR revealed that JDCaP-hr retained wild type AR. AR mRNA expression in JDCaP-hr was approximately 7-fold higher than in JDCaP, and interestingly, truncated AR protein with molecular weight of approximately 75 kDa in addition to full length AR (110 kDa) was expressed in JDCaP-hr. Microarray analysis revealed that ubiquitin-dependent protein catabolism pathway was markedly upregulated in JDCaP-hr compared with JDCaP. JDCaP-hr cells proliferated in vitro in steroid-free medium under co-culture condition with mouse stromal cells, and secreted prostate-specific antigen into the conditioned medium. Taken together, JDCaP/JDCaP-hr is a valuable model which simulates the recurrence of ADPC to castration-resistant prostate cancer (CRPC) with common clinical features such as AR overexpression. This model provides not only useful in vivo and in vitro systems to evaluate anticancer agents for ADPC and CRPC but also intriguing implications for the recurrence of ADPC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3247.

Gene expression during inactivity-induced muscle atrophy: effects of brief bouts of a forceful contraction countermeasure

Anabolic and catabolic markers of muscle protein metabolism were examined in inactivity-induced atrophying muscles with and without daily short-duration, high-resistance isometric contractions. Inactivity was achieved via spinal cord isolation (SI), which results in near inactivity of the hindlimb musculature without compromising the motoneuron-muscle connectivity. Adult rats were assigned to a control (Con) or SI group in which one limb was stimulated (SI-Stim, 5 consecutive days of brief bouts of high-load isometric contractions) while the other served as a SI control (SI). Both the medial gastrocnemius (MG) and soleus weights (relative to body weight) were approximately 71% of Con in the SI, but maintained at Con in the SI-Stim group. Activity of the IGF-1/phosphatidylinositol 3-kinase (PI3K)/Akt pathway of protein synthesis was similar among all groups in the MG. Expression of atrogin-1 and muscle RING finger-1 (MuRF-1), markers of protein degradation, were higher in the MG and soleus of the SI than Con and maintained at Con in the SI-Stim. Compared with Con, the anti-growth factor myostatin was unaffected in the MG and soleus in the SI but was lower in the MG of the SI-Stim. These results demonstrate that upregulation of specific protein catabolic pathways plays a critical role in SI-induced atrophy, while this response was blunted by 4 min of daily high-resistance electromechanical stimulation and was able to preserve most of the muscle mass. Although the protein anabolic pathway (IGF-1/PI3K/Akt) appears to play a minor role in regulating mass in the SI model, increased translational capacity may have contributed to mass preservation in response to isometric contractions.

Effect of starvation on global gene expression and proteolysis in rainbow trout (Oncorhynchus mykiss).

BackgroundFast, efficiently growing animals have increased protein synthesis and/or reduced protein degradation relative to slow, inefficiently growing animals. Consequently, minimizing the energetic cost of protein turnover is a strategic goal for enhancing animal growth. Characterization of gene expression profiles associated with protein turnover would allow us to identify genes that could potentially be used as molecular biomarkers to select for germplasm with improved protein accretion.ResultsWe evaluated changes in hepatic global gene expression in response to 3-week starvation in rainbow trout (Oncorhynchus mykiss). Microarray analysis revealed a coordinated, down-regulated expression of protein biosynthesis genes in starved fish. In addition, the expression of genes involved in lipid metabolism/transport, aerobic respiration, blood functions and immune response were decreased in response to starvation. However, the microarray approach did not show a significant increase of gene expression in protein catabolic pathways. Further studies, using real-time PCR and enzyme activity assays, were performed to investigate the expression of genes involved in the major proteolytic pathways including calpains, the multi-catalytic proteasome and cathepsins. Starvation reduced mRNA expression of the calpain inhibitor, calpastatin long isoform (CAST-L), with a subsequent increase in the calpain catalytic activity. In addition, starvation caused a slight but significant increase in 20S proteasome activity without affecting mRNA levels of the proteasome genes. Neither the mRNA levels nor the activities of cathepsin D and L were affected by starvation.ConclusionThese results suggest a significant role of calpain and 20S proteasome pathways in protein mobilization as a source of energy during fasting and a potential association of the CAST-L gene with fish protein accretion.

Proteasome inhibitor MG-132 enhances whole-body protein turnover in rat

Proteasome inhibitors are novel therapeutic agents which may be used in treatment of cancer and other severe disorders. We studied the effect of proteasome inhibitor MG-132 on protein and amino acid metabolism. In MG-132-treated rats we observed a significant decrease in proteasome-dependent proteolysis in skeletal muscle and an increase in whole-body protein turnover (i.e., increase in whole-body proteolysis and protein synthesis). Proteasome-dependent proteolysis was activated in the liver and kidney, protein synthesis increased in skeletal muscle, liver, and kidney. Insignificant changes were found in jejunum and colon. MG-132 administration induced a significant increase in concentration of several amino acids in blood plasma and their decrease in jejunum and colon. We conclude that administration of MG-132 affects both protein anabolic and protein catabolic pathways via the direct effect on proteasome-dependent proteolysis and indirect effect on proteolysis and protein synthesis via unidentified mediators.

The Role of Tricorn Protease and Its Aminopeptidase-Interacting Factors in Cellular Protein Degradation

Tricorn protease was previously described as the core enzyme of a modular proteolytic system displaying multicatalytic activity. Here we elucidate the mode of cooperation between Tricorn and its interacting factors, and we identify two additional factors, F2 and F3, closely related aminopeptidases of 89 kDa. In conjunction with these three factors, Tricorn degrades oligopeptides in a sequential manner, yielding free amino acids. We have been able to reconstitute a proteolytic pathway comprising the proteasome, Tricorn, and its interacting factors, F1, F2, and F3, which converts proteins efficiently into amino acids. Therefore, it is quite likely that Tricorn also acts in vivo downstream of the proteasome and, in cooperation with its interacting factors, completes protein catabolic pathways.

Chapter 3 Ubiquitin and its role in neurodegeneration

The major human neurodegenerative diseases are often characterized by the accumulation of abnormal proteins, either intracellularly in the form of intraneuronal inclusion bodies or extracellularly as amyloid plaques. Both of these pathological structures may be a result of altered protein catabolic and/or processing pathways. The involvement of one of the major intracellular protein catabolic routes, the ubiquitin/proteasome pathway, and its possible role in the development of neurodegenerative disease is discussed in this chapter. Ubiquitin is a compact (8.5 kDa) molecule that is ligated post-translationally to a variety of cytosolic and membrane proteins in an ATP-dependent process involving the formation of an isopeptide bond between the C-terminal glycine residue of ubiquitin and an ɛ-amino group of a lysine residue of the protein substrate. The formation of ubiquitinated proteins requires a multienzyme process, which is still not fully understood. Ubiquitin is first activated by a ubiquitin-activating enzyme (E 1) and is then transferred to a ubiquitin-conjugating enzyme (E2).

Persistent protein damage despite reduced oxygen radical formation in the aging rat brain

The relation between cerebral oxygen radicals and the aging process was investigated in crude synaptosomal (P2) fractions from rats. The rate of formation of oxygen radicals was measured using the probe 2′,7′-dichlorofluorescein diacetate (DCFH-DA), which is de-esterified and subsequently oxidized by oxygen radicals to a fluorescent product 2′,7′-dichlorofluorescein (DCF). There was a significant agedependent decrease in the formation rate of oxygen radicals, observed by decreased formation of DCF. No difference in oxygen radical formation was apparent between age groups following an in vitro challenge with an ascorbate/FeSO4 mixture. This age-dependent decrease in cerebral oxygen radical generation coincided with age-dependent increases in Superoxide dismutase. No age-related alterations in lipid order in either the hydrophilic or lipophilic membrane regions were observed using fluorescence polarization analysis. Age-dependent losses in cerebral P2 tryptophan fluorescence (a measure of protein degradation), and increased liberation of [14C]protein fragments into the acid-soluble fraction (a measure of overall proteolytic activity) were observed. Results suggest that aging does not proceed as a result of elevated rates of generation of oxygen radicals, a finding that does not support the proposed free radical theory of aging. The observed age-dependent decrease in the formation of oxygen radicals does not effect membrane lipid order. These findings implicate modifications in proteins and activated protein catabolic pathways as major contributing factors in the normal physiological process of senescence.