[Changes in the Activities and Contents of Individual Forms of Proteasomes in Samples of the Cerebral Cortex during Pathology Development in 5xFAD Mice].

The ubiquitin-proteasome system (UPS) provides hydrolysis of most intracellular proteins in proteasomes. There are various forms of proteasomes that differ, among other things, in the set of proteolytic subunits and the presence of activators. Alzheimer's disease (AD) is characterized by disturbances in the functional state of the UPS. At the same time, an increase in the expression of certain forms of proteasomes, in particular, proteasomes containing immune subunits (nonconstitutive proteasomes), has been shown. Here, we studied dynamic changes in the expression of catalytic proteasome subunit genes and corresponding proteins in the cerebral cortex of animals using a mouse model of AD (5xFAD transgenic mice). Increases by 4 and 6 folds in transcripts of the PSMB9 and PSMB8 genes encoding immune proteasome subunits were detected, as well as a significant increase in the content of immune ß-subunits (by 2.8 folds, ß1i; 2.2 folds, ß2i) in samples from 5xFAD mice at the age of 380 days, compared with samples from mice at 60 days of age. Moreover, the activation of both 20S and 26S proteasomes containing immune subunits were revealed in samples from 380 days old 5xFAD mice by electrophoresis in native conditions. This indicates activated synthesis of the immune subunits and assembly of nonconstitutive proteasomes at the terminal stage of pathology development. The obtained data, in combination with the available literature, indicate that the activation of nonconstitutive proteasomes is a universal phenomenon characteristic of various animal models of AD, which may reflect both the development of neuroinflammation and adaptive processes in tissues induced by the accumulation of toxic protein aggegates.

[Dynamic Changes in the Activities and Contents of Particular Proteasome Forms in the Cerebral Cortex of C57BL/6 Mice during Aging].

Proteasomes are key components of the ubiquitin-proteasome system. Various forms of proteasomes are known. During aging, disturbances in the functioning of proteasomes have been revealed, as well as increased expression of their particular forms. Considering these data, we studied the expression of genes encoding the constitutive and immune subunits of proteasomes in cerebral cortex samples from C57BL/6 mice at the ages of 60, 190, 380, and 720 days. In addition, the contents of constitutive and immune proteasome subunits, chymotrypsin-like and caspase-like activities of proteasome pools, as well as the activity of the ß5i immune subunit were studied in tissue homogenates. The chymotrypsin-like activity and the activity of the ß5i subunit of different forms of proteasomes separated by electrophoresis in native gel were characterized. Compared with samples from young animals, in the cerebral cortex of animals at an age of 720 days the following changes in the expression patterns of proteasome genes were revealed: a decreased expression of the PSMB5 gene encoding constitutive proteasome subunit ß5; increased expression of genes encoding immune proteasome subunits ß5i and ß1i. In tissue homogenates of aged mice, an increase in the content of immune subunits ß1i and ß2i was shown. In samples from old animals, chymotrypsin-like activity was decreased and a tendency to a decrease in caspase-like activity of proteasomes as well as the ß5i subunit activity was revealed. Analysis of the activity of native complexes in tissues obtained from old animals revealed decreased chymotrypsin-like activity of 26S and 20S proteasomes containing the ß5i subunit. Based on the obtained data, it can be assumed that changes in the pool of nonconstitutive proteasomes reflect aging-associated adaptive processes in the mouse brain.

Native structure of rat liver immune proteasomes.

Native structure of active forms of rat liver immune proteasomes has been studied by two-dimensional electrophoresis method modified for analysis of unpurified protein fractions. The developed method allowed revealing the proteasome immune subunits LMP7 and LMP2 in 20S subparticles and in the structures bound to one or two PA28αß activators, but not to the PA700 activator, which is involved in the hydrolysis of ubiquitinated proteins. The results obtained indicate the participation of the immune proteasomes in delicate regulatory mechanisms based on the production of biologically active peptides and exclude their participation in processes of crude degradation of "rotated" ubiquitinated proteins.

Role of proteasomes in non-specific immune response of marine annelids.

We investigated functioning of proteasomes and chaperones in Arenicola marina coelomocytes in conditions of lipopolysaccharide-induced inflammation. We observed the increase of chymotrypsin-like proteasome activity in coelomocytes 1 h after induction. Amount of proteasome subunits alpha- and beta-5 types increased as well. We also detected appearance of a new form of Hsp70 chaperone in infected coelomocytes. Our results allow us to consider the changes in proteasome structure and induction of chaperones as principle mechanisms in stress adaptation and defensive reactions development in annelids.

Changes in proteasome chymotrypsin-like activity during the development of human mammary and thyroid carcinomas.

Changes in the proteasome chymotrypsin-like activity in mammary and thyroid carcinomas in comparison with the adjacent tissue were studied at stages T(1-4)N(0-3)M(0) and T(2-3)N(0-1)M(0), respectively. The activities changed in a wave-like manner over the course of mammary carcinoma growth in cases with and without metastases. The minimum increment of the activity in the tumor was recorded during the T(2)N(0) stage in the absence of local metastases. The increment of the activity reached the peak in N(1) tumors of the same size with metastases. The activities in the tumor and adjacent tissues virtually did not differ during the T(3-4)N(1-3) stages. The time course of proteasome activity changes in thyroid tumors of the studied stages was similar to that in mammary carcinoma. The results can be used for development of methods for evaluating the aggressiveness of mammary and thyroid tumors.

[Immune proteasomes in the development of rat immune system].

The dynamics of the expression of LMP7 and LMP2 proteasome subunits in embryonic and early postnatal development of rat spleen and liver is investigated in comparison with the dynamics of chymotrypsin-like and caspase-like proteasome activities and expression of MHC (major histocompatibility complex) class I molecules. The immune subunits LMP7 and LMP2 distribution in spleen and liver cells in the development process is also studied. A mutual for both organs tendency to the increase of the expression of both LMP7 subunit and LMP2 one on P21 (the 21st postnatal day) as compared to the embryonic period is discovered. However, the total proteasome level is shown to be constant. At definite development stages, the dynamics of immune subunits expression in the spleen and liver was different. In the spleen gradual enhancement of both immune subunits level being detected on P1, P18 and P21, in the liver gradual enhancement periods on E16 (the 16th embryonic day) and E18 changed to the stage of the shrink of immune subunits level on P5. This level did not reliably change till P18 and was augmented on P21. The alterations revealed were accompanied by chymotrypsin-like activity raise and caspase-like activity drop in spleen by P21 as compared with the embryonic period, which proves the enlargement of proteasome ability to form antigenic epitopes for MHC class I molecules. In the liver, both activities increased by P21 in comparison with the embryonic period. Such dynamics of caspase-like activity can be explained not only by the change of proteolytic constitutive and immune subunits, but also by additional regulatory mechanisms. Besides, it is discovered that the increment of immune subunits expression in the early spleen development is connected with the process of successive forming the white pulp by B- and T-lymphocytes enriched by immune subunits. In the liver, the growth of immune subunits level by P21 was accompanied by their expression expansion in hepatocytes, while their plunge by P5 may be related to the loss of liver function of a primary lymphoid organ of the immune system by this stage and disappearance of B-lymphocytes enriched by immune proteasomes in it. In the spleen and liver, MHC class I molecules were revealed at the periods of the raise of proteasome immune subunits level. On E21 , the liver was enriched by neuronal NO-synthase, its level decreased after birth and enhanced to P18. This fact indicates the possibility of the induction of the immune subunits LMP7 [character: see text] LMP2 expression in hepatocytes in signal way with neuronal NO-synthase participation. The results obtained prove that T-cell immune response with spleen participation as regards rat liver cells is possible starting with P19-P21 stage. First, at this period, white pulp T-area is formed in the spleen. Second, enhanced immune proteasomes and MHC class I molecules levels in hepatocytes can procure antigenic epitopes formation from foreign proteins and their delivery to cell surface for their subsequent presentation for cytotoxic T-lymphocytes.

Peculiarities of proteasome pool formation in rat spleen and liver during postnatal development.

Changes in the specific activity and amounts of 26S and 20S proteasome pools in rat spleen and liver during postnatal development and appearance in them of immune subunits were studied. Two decreases in chymotrypsin-like activity of the proteasome pools were recorded during the first three weeks after birth. The activity minimum fell on the 11th and 19th days, and the first decrease was more prolonged and pronounced than the second. The decrease in the specific activity of the 26S proteasome pools was associated with a reduction of their quantity. The 20S proteasome pools displayed no such decreases. Noticeable quantities of immune subunits LMP7 and LMP2 were revealed by Western blotting in the spleen on the 7th day and on the 19th day in the liver, concurrently with the beginning of the decrease in the proteasome activity. It was concluded that during the first three weeks of postnatal development the proteasome pools in rat spleen and liver were replaced twice, and in the spleen (a lymphoid organ) a qualitatively new pool containing immune subunits appeared nearly two weeks earlier than in the liver (a non-lymphoid organ). The appearance of immune proteasomes in different organs and tissues during some weeks after birth seems to explain the immune system inefficiency during embryogenesis and early postnatal development.

[Multiple forms of the proteasomes and some approaches to their separation]. / Mnozhestvennost' form proteasomy i nekotorye podkhody k ikh razdeleniiu.

A technique for proteasome isolation from rat liver was developed; it allows isolation of 26S proteasome fraction free from 20S proteasome admixture. The technique includes consecutive stages of protein fractionation by different concentrations of ammonium sulfate, gel filtration on Sepharose 2B, and high-speed ultracentrifugation.

Endogenous factor activating Na,K-ATPase induced by blockade of adrenoceptors.

It was established that the regulatory factor released from the effector cells under the blockade of postsynaptic alpha- and beta-adrenoceptors by phentolamine and propranolol (0.5-1.0 microM) and activating noradrenergic uptake and synthesis increased the activity of Na,K-ATPase of isolated rat organs and partially purified Na,K-ATPase isolated from the rat brain. The regulatory factor is a protein with molecular mass 25-100 kDa, adsorbed on phenylsepharose CL-4B and eluated with 30 mM KCl. It consists of two active components with pI 5.1 and 5.9 which in admixture (1:1) activate Na,K-ATPase by 60%.

[Changes in Na, K-ATPase activity following adrenoreceptor blockade]. / Izmenenie aktivnosti Na, K-ATFazy pri blokade adrenoretseptorov.

A regulatory factor released from the effector cell during blockade of postsynaptic alpha- and beta-adrenoreceptors with phentolamine and propranolol (5 X 10(7)-1 X 10(-6) M) and activating norepinephrine neuronal uptake and synthesis, was shown to activate, Na, K-ATPase of the rat isolated organs as well as the preparation of partly purified Na, K-ATPase extracted from the rat brain. Ouabain (1 X 10(-3) M) eliminated completely the activating effect of the regulatory factor on the ATPase.