Lysenko La

Biochemical markers of pollutant responses in macrozoobenthos from the White Sea: Intracellular proteolysis

Coastal environments of Kandalaksha Gulf in the White Sea (Russia) despite nature conservation efforts are heavily influenced by human activities. Biological effects of complex environmental pollution, including organic substances, heavy metals, and oil hydrocarbons, were assessed in widely distributed marine invertebrates, Gammarus duebeni (Crustacea, Amphipoda) and Mytilus edulis (Mollusca, Bivalvia), collected from a series of anthropogenically-impacted areas and distanced reference sites in Kandalaksha Gulf. The parameters of intracellular protein degradation pathways such as cytosol calpain system and lysosomal cathepsins B (CatB) and cathepsin D (CatD) were studied. The response reactions observed in invertebrates vary in specificity and ranged from adaptive to destructive depending on the total contaminant level and the nature of predominant pollutant. The ecological relevance of studied parameters as biomarkers was confirmed by their ability to indicate both expose to pollutants and adverse effects at the organism level.

Effect of copper and cadmium ions on heart function and calpain activity in blue mussel Mytilus edulis

The heart rate and calpain activity of blue mussels Mytilus edulis from the sublittoral zone, exposed to different levels of water-borne copper and cadmium, was investigated in a long-term experiment. The content of cadmium and copper in the blue mussel was determined using flame and graphite Atomic absorption spectroscopy. The observed concentrations ranged from 2.5 to 89.1 μg/g dry weight for cadmium and from 6.1 to 51.0 μg/g dry weight for copper in the control and highest concentration, respectively. Initially, increase in cardiac activity in response to copper and Cadmium exposure was observed under all pollutant concentrations (5–250 and 10–500 μg/L, respectively). The calpain-like activity in gills and hepatopancreas of the mussels treated with metals changed in dose- and time-dependent manner: from a sharp rise at the 250 μg/L concentration of copper on the first day to a significant decrease under the effect of Cadmium in the concentration of 500 μg/L on the third day of the experiment. These results suggest that: (i) heart rate oscillation may reflect active adaptation of blue mussels to contamination and (ii) animals have different sensitivity to copper and Cadmium according to the role of the metals in the mussels’ life activity.

Calpain system dysregulation in rat brain at beta-amyloid-induced neurodegeneration

Experimental evidence of calcium-dependent proteolysis dysregulation in brain of the murine model of Alzheimer’s disease was obtained. Experimental treatment, consisting of intra-hippocampal injection of amyloid beta-peptide (Aβ1–40), promoted activation of main calpain forms in murine brain along with a decrease in the content of a natural calpain inhibitor, calpastatin. As a result of a prognostic experiment on the correction of neurodegeneration induced in rats, neuroprotective properties of a steroid hormone estradiol were confirmed and a possible mechanism of the protective effect was suggested. The results allow consideration of both biochemical modifications in protein facilities of a pathology-affected brain and the mechanisms of neurodegeneration and neuroprotection.

The basal calcium level in fibers of the rat soleus muscle under gravitational unloading: The mechanisms of its increase and the role in calpain activation

The first data on increase in the basal level of cal� cium in immobilized muscles appeared in 1970 (3). Nasledov and coworkers hypothesized that calcium ions accumulated under the conditions of gravita� tional unloading (1). In that study, the authors used the model of antiorthostatic suspension. Using an isolated rat m. soleus, the mechanical response has been dem� onstrated to attenuate rapidly after a series of tetanic contractions in calciumfree Ringers solution. How� ever, after 14�day unloading in the standard model of antiorthostatic suspension, there was no attenuation in tests performed on muscles of the suspended ani� mals. When, in the second experimental series, Vera� pamil (a calcium channel blocker) was added to the calciumfree medium, attenuation of the mechanical response was observed in the suspended animals. It was suggested that an increased resistance to muscle fatigue in calciumfree Ringers solution was a result of intracellular accumulation of calcium ions, which freely came out of the muscle through the channels, and Verapamil inhibited this process. Using the calcium probe Fluo�3, a significant increase in the concentration of calcium ions of the mouse m. soleus was later demonstrated as soon as the second day of suspension (8). An excessive amount of calcium ions might occur under microgravity in the myoplasm of the muscle fiber either from the extracellular space through exter� nal calcium channels or from the sarcoplasm reticu� lum because of leakage of Ryanodine channels. There is evidence suggesting that Ryanodine channels are involved in increasing the intracellular calcium (12). In addition, a higher expression of dihydropyri� dine receptors was already observed upon shortterm gravitational unloading (9). It was subsequently dem� onstrated that the use of Nifedipine, a selective blocker of calcium channels, against the background of 14�day functional unloading led to a significant diminishing the level of calcium ions in the myoplasm of m. soleus fibers, and it largely prevented the trans� formation of the myosin phenotype (6, 12). At the same time, peroral Nifedipine administration may ini� tiate multiple systemic effects. Because of this, only a decrease in the basal level of calcium ions in the mus� cle fiber in response to a direct Nifedipine action against the background of gravitational unloading can serve as valid evidence of the involvement of slow Ltype calcium channels into accumulation of these ions in fibers of postural muscles. An increase in the calcium ion concentration in the myoplasm of muscular fibers at rest may stimulate the activity of calpains, calciumdependent proteases. Both µ� and mcalpains, which are activated by microand millimolar calcium concentrations, respectively, are present in all tissues of vertebrate ani� mals. These enzymes play the key role in the degrada� tion of a series of cytoskeletal proteins (2). The calpain activity has been demonstrated to grow up signifi� cantly even upon a shortterm gravitational unloading (4, 5), and this was accompanied by a significant decrease in the concentration of desmin, one of the key cytoskeletal muscle proteins. At the same time, the importance for increasing the calpainstimulating activity of calcium ions in the muscle fibers during gravitational unloading, may be understood after application of calcium chelating agents with an intra� cellular activity under these conditions.

The effect of weak low-frequency magnetic fields on the intracellular calcium-dependent proteinases of fish

The in vivo and in vitro effects of weak, low-frequency magnetic fields with resonance parameters for calcium ions upon intracellular calcium-dependent proteinases (calpains) in the crucian carp (Carassiuscarassius (L.)) and roach (Rutilus rutilus (L.)) were studied. It has been revealed that the impact of a weak lowfrequency magnetic field leads to considerable decrease in the activity of calpains in the fish skeletal muscles and brain.

Protein degradation systems in the skeletal muscles of parr and smolt Atlantic salmon Salmo salar L. and brown trout Salmo trutta L.

Although protein degradation limits the rate of muscle growth in fish, the role of proteolytic systems responsible for degrading myofibrillar proteins in skeletal muscle is not well defined. The study herein aims to evaluate the role of calpains (calcium-activated proteases) and proteasomes (ATP-dependent proteases) in mediating muscle protein turnover at different life stages in wild salmonids. Protease activities were estimated in Atlantic salmon (Salmo salar L.) and brown trout (Salmo trutta L.) parr and smolts from the Indera River (Kola Peninsula, Russia). Calpain and proteasome activities in Atlantic salmon skeletal muscles were lower in smolts as compared with parr. Reduced muscle protein degradation accompanying Atlantic salmon parr-smolt transformation appeared to provide intense muscle growth essential for a minimum threshold size achievement that is required for smoltification. Calpain and proteasome activities in brown trout parr and smolts at age 3+ did not significantly differ. However, calpain activity was higher in smolts brown trout 4+ as compared with parr, while proteasome activity was lower. Results suggest that brown trout smoltification does not correspond with intense muscle growth and is more facultative and plastic in comparison with Atlantic salmon smoltification. Obtained data on muscle protein degradation capacity as well as length-weight parameters of fish reflect differences between salmon and trout in growth and smoltification strategies.

Skeletal muscle protease activities in the early growth and development of wild Atlantic salmon (Salmo salar L.)

Growth-related dynamics of intracellular protease activities in four year classes of the Atlantic salmon (Salmo salar L. 1758) parr and smolts inhabiting salmon rivers of northwestern Russia (the White Sea basin) were studied. Cathepsin B, cathepsin D, proteasome, and calpain activities in the skeletal muscles of salmon were assessed to investigate their relative contribution to the total protein degradation as well as to young fish growth process. It was confirmed that calpain activity dominates in salmon muscles while proteasome plays a minor role, in contrast to terrestrial vertebrates. Calpain and proteasome activities were maximal at the early post-larval stage (in parrs 0+) and declined with age (parrs 1+ through 2+) dropping to the lowest level in salmon smolts. Annual growth increments and proteolytic activities of calpains and proteasome in the muscles of salmon juveniles changed with age in an orchestrated manner, while lysosomal cathepsin activities increased with age. Comparing protease activities and growth increments in salmon parr and smolts we suggested that the partial suppression of the protein degradation could be a mechanism stimulating efficient growth in smoltifying salmon. Growth and smoltification-related dynamics of protease activities was quite similar in salmon populations from studied spawning rivers, such as Varzuga and Indera; however, some habitat-related differences were observed. Growth increments and protease activities varied in salmon parr 0+ (but not on later ages) inhabiting either main rivers or small tributaries apparently due to habitat difference on the resources for fish growth.

The role of lysosomal proteinases and estradiol in neurodegeneration induced by beta-amyloid

Activation of lysosomal degradation proceßs in nervous tißsue may be neuroprotective. One of the factors that may influence on expreßsion of lysosomal proteinases is the sex hormone, estradiol (E2). In this regard the expreßsion of lysosomal proteinases after intracerebral injection of beta-amyloid peptide (Aβ) was investigated as well as the neuroprotective effect of E2 in Aβ-induced neurodegeneration. Intracerebral injection of Aβ was shown to cause the significant increase in expression of cathepsin D in rat hippocampus and cerebral cortex. On the background of Aβ intoxication, E2 treatment resulted in further increase in cathepsin D gene expression in hippocampus region and in its lowering to the control level in cerebral cortex. It was demonstrated for the first time that neuroprotective effect of E2 may be mediated by cathepsin D up-regulation.

Molecular Evolution Within Protease Family C2, or Calpains

Calpains, or the intracellular Ca2+-dependent proteases (EC 3.4.22.17, family C2, cysteine protease clan CA) are one of the most important proteolytic systems in cytosol of eukaryotic and some prokaryotic cells (reviews: Croall & DeMartino, 1991; Goll et al., 2003; Sorimachi et al., 2011a). Calpain family is ancient and diverse since highly variable modules flanking conservative proteolytic core are found in the structure of these proteins. Linking of protease catalytic domain with ancillary domains, i.e. specialized functional modules with their own spectrum of non-proteolytic activities and binding partners, expands calpain functions on multiple cellular processes. Calpains are processing proteases cleaving their specific substrates at one or a limited number of sites to modulate their structure and activity rather than degrade it. Calpains are versatile proteases that have been implicated in diverse cellular signaling pathways mediated by calcium, such as cytoskeleton remodeling, cell-cycle regulation, differentiation, and death (Croall & DeMartino, 1991; Carafoli & Molinari, 1998; Goll et al., 2003; Nemova et al., 2010). Calpains were also considered to participate in chromosome rearrangements during mitosis (Schollmeyer, 1988), microtubule assembly and disassembly (Billger et al., 1988), intracellular signaling, motility, and vesicle traffic (Choi et al., 1997; Huttenlocher et al., 1997; Lu et al., 2002; Li & Iyengar, 2002). In mammals and plants it is clear that calpains are of critical importance for development (Wang et al., 2003; Dutt et al., 2006; Sorimachi et al., 2010). Furthermore, impaired calpain activity due to mutations or misregulation of the calpains has been implicated in a variety of pathological conditions including muscular dystrophy, ischemia, diabetes mellitus, cancer, and neurodegenerative disease (Tidball & Spencer, 2000; Huang & Wang, 2001; Crocker et al., 2003; Mamoune et al., 2003; Suzuki et al., 2004; Zatz & Starling, 2005). Calpains have attracted much attention because of the recent discovery of correlations between calpain gene mutations and human diseases, together with elucidation of its three-dimensional structure (Hosfield et al., 1999; Strobl et al., 2000) and Ca2+-induced activation mechanisms (Moldoveanu et al., 2002; 2004). Because the enzyme participates not only in normal intracellular signal transduction cascades but also in various pathological states, calpain research has attracted tremendous interest in wide areas of life sciences in both basic and clinical terms.