[Progerin and Its Role in Accelerated and Natural Aging].

Well-known theories of aging suggest that a certain metabolic defect negatively affects vital activity of the cell, be it oxidative stress, the accumulation of lesions in DNA, the exhaustion of telomeres, or distorted epigenetic processes. The theory of aging considered in the review postulates that an accumulation of progerin on the inner side of the nuclear envelope underlies the above defects. Progerin is a defective precursor of the lamin A nuclear matrix protein in which the C-terminal cysteine, which is removed normally, is retained and modified with a hydrophobic oligoisoprene chain. Progerin molecules attach with their hydrophobic processes to the inner membrane of the nuclear envelope, pushing away the adjacent fibrils of the nuclear matrix and the chromatin periphery. This changes the morphology and shape of the nucleus and alters the properties of the nuclear envelope and pore complexes embedded in it. As progerin accumulates in the nucleus, structural distortions increase in the nucleus, further distorting the nuclear-cytoplasmic transport of macromolecules and leading to the above defects in cell metabolism. This leads to increasing cell death and aging of the body over time. This mechanism of aging has been identified in patients with Hutchinson-Gilford progeria syndrome (HGPS). Mass progerin production in HGPS is caused by the point mutation c.1824C→T in exon 11 of the LMNA gene, which codes for lamins A and C. The mutation stimulates non-standard splicing of the primary transcript during the formation of the lamin A precursor mRNA, thus causing progerin production. Children with progeria who have received the mutation from one of their parents age rapidly and die before 15 years of age. Approaches to progeria treatment are aimed at preventing the formation of progerin or destroying the progerin that has already accumulated. In the latter case, a promising strategy is to use rapamycin or its analogs and other substances and techniques that activate autophagy to purify the cell from progerin. Although in much smaller amounts, progerin is found in progeria-free people and may therefore play a role in natural aging. A maximum age that a person can reach is possible to estimate by taking account of the role that progerin plays in telomere shortening. Encouraging preliminary results achieved in purifying cells from progerin provide a means to develop an optimal procedure for periodic purification of the human body from progerin in order to reduce the rate of aging.

On the activation of calcium-dependent proteolysis in brain neurons of spontaneously hypertensive rats (SHR strain).

Females of spontaneously hypertensive (SHR strain) and normotensive rats (WKY strain and Wistar) received drinking water with normal (80 mg/liter) or reduced concentration of Ca(2+)(8 mg/liter). Activity of calcium-dependent calpain protease in neurons did not differ in 18-day-old rat pups born and suckled by these animals. Our results are consistent with published data on normal metabolism of SHR rats up to the age of 30 days.

Specific proteolysis of neuronal protein GAP-43 by calpain: characterization, regulation, and physiological role.

The mechanism of specific proteolysis of the neuronal protein GAP-43 in axonal terminals has been investigated. In synaptic terminals in vivo and in synaptosomes in vitro GAP-43 is cleaved only at the single peptide bond formed by Ser41; this is within the main effector domain of GAP-43. Proteolysis at this site involves the cysteine calcium-dependent neutral protease calpain. The following experimental evidences support this conclusion: 1) calcium-dependent proteolysis of GAP-43 in synaptosomes is insensitive to selective inhibitor of micro-calpain (PD151746), but it is completely blocked by micro- and m-calpain inhibitor PD150606; 2) GAP-43 proteolysis in the calcium ionophore A23187-treated synaptosomes is activated by millimolar concentration of calcium ions; 3) the pattern of fragmentation of purified GAP-43 by m-calpain (but not by micro-calpain) is identical to that observed in synaptic terminals in vivo. GAP-43 phosphorylated at Ser41 by protein kinase C (PKC) is resistant to the cleavage by calpain. In addition, calmodulin binding to GAP-43 decreases the rate of calpain-mediated GAP-43 proteolysis. Our results indicate that m-calpain-mediated GAP-43 proteolysis regulated by PKC and calmodulin is of physiological relevance, particularly in axonal growth cone guidance. We suggest that the function of the N-terminal fragment of GAP-43 (residues 1-40) formed during cleavage by m-calpain consists in activation of neuronal heterotrimeric GTP-binding protein G(o); this results in growth cone turning in response to repulsive signals.

Site-specific calcium-dependent proteolysis of neuronal protein GAP-43.

GAP-43 is a presynaptic protein participating in signal transduction processes in nerve terminals. GAP-43 exists in neurons along with two truncated forms devoid of 4 and 40 N-terminal residues. In this report, we show that these forms of GAP-43 are proteolytic fragments derived from calcium-dependent cleavage of GAP-43 molecule at 5th and 41st residues. GAP-43 site-specific proteolysis in synaptosome and cytosol fractions proved to be dependent on the addition of millimolar amounts of calcium. This fact together with inhibition of GAP-43 proteolysis by calpain inhibitors as well as local composition of the cleavage sites indicates to the participation of calpain in this process. The proteolysis disturbs some properties characteristic for whole GAP-43 molecules, in particular, calmodulin binding and Ser-41 phosphorylation, when the cleavage occurs at 41st residue. Some other GAP-43 properties (G(o) protein activation and membrane attachment) are retained by separate fragments. Therefore, calcium controlled site-specific proteolysis of GAP-43 can be of great physiological significance.

Not growth associated protein GAP-43 (B-50), but its fragment GAP-43-3 (B-60) predominates in rat brain during development.

The participation of the nerve termini growth associated protein GAP-43 in neurite outgrowth and targeting is well documented. Commonly, besides GAP-43 itself, two big fragments devoid of four (GAP-43-2, IB-50) and of about 40 (GAP-43-3, B-60) N-terminal residues were co-isolated from brain. In adult brain, GAP-43 significantly prevails over the fragments. To find their relative amounts during development, rat brain proteins were isolated on different stages of embryonal and post-natal development and subjected to gel electrophoresis in 0.9 M acetic acid-2.5 M urea system. The bands of GAP-43 protein family were detected on Western blots. We show that in developing brain (until 5th post-natal day), a proteolysis of GAP-43 near Ser(41) that results in GAP-43-3 accumulation is activated. We hypothesize that just the functions that can be performed by the GAP-43 fragments are of importance for developing brain.

Enhanced level of site-specific proteolysis of GAP-43 protein during early stages of brain development.

GAP-43 protein of nerve terminals (B-50, F1, F57, pp46, neuromodulin) is thought to be one of key proteins involved in the control of outgrowth of neurites, release of neuromediators, synapse plasticity, etc. GAP-43 is usually considered as a whole protein. Along with the intact protein, nerve cells also contain two large native fragments of GAP-43 deprived of four or of about forty N-terminal amino acid residues (GAP-43-2 and GAP-43-3, respectively). The full-length GAP-43 is predominant in the mature brain. However, the ratio of the full-length protein and its fragments can vary under different physiological conditions. Changes in the GAP-43 proteins (the full-length protein and its fragments) were studied during embryonal and postnatal development of rat brain. The GAP-43 proteins were found to be expressed not later than on the 12-13th day of embryogenesis. Then their contents increased, and, until the 10th day after birth, GAP-43-3 dominated rather than the full-length protein. It is suggested that during this period the activity of a specific protease, which cleaves the N-terminal peptide of about 40 residues from the full-length GAP-43 molecule, is increased. The cleavage occurs in the region responsible for the interaction of GAP-43 with calmodulin. In the full-length molecule, this region is responsible also for the recognition of Ser41 residue by protein kinase C during phosphorylation. Another functionally important region that determines, in particular, the attachment of GAP-43 to the plasma membrane is cleaved from the main part of the molecule together with the N-terminal peptide. Thus, the specific fragmentation of GAP-43 that depends on developmental stage should be considered as a controlled structural rearrangement fundamentally affecting the functions of this protein.

The BASP1 family of myristoylated proteins abundant in axonal termini. Primary structure analysis and physico-chemical properties.

Proteins BASP1 and GAP-43/B-50, which are abundant in nerve endings, show a number of similar physico-chemical properties. Nevertheless, they belong to different protein families. In this work, complete amino acid sequences of bovine BASP1 and human BASP1 were established. They proved to be very similar to the sequences of rat brain protein NAP-22 and chicken brain protein CAP-23. Relatively to human BASP1 its bovine, rat and chicken analogues show 80%, 70% and 45% sequence identity respectively, confirming their membership of a definite protein family (BASP1 family). All members of BASP1 family contain several 'good' PEST sequences characteristic for short-living proteins. Conservation of PEST sequences in BASP1 of different species points to their significance for BASP1 functions. In contrast to GAP-43/B-50 showing high immunological cross-reactivity between the proteins belonging to different species of mammals, immunological properties of BASP1 are species specific. BASP1 shows both high hydrophilicity and some properties characteristic for hydrophobic proteins. These properties are caused by N-terminal myristoylation of BASP1 molecules. Unlike GAP-43/B-50, BASP1 is present in high amounts also in some non-nervous tissues: testis, kidney and lymphoid organs (spleen, thymus). So far examined characteristics, including myristoylation, peptide maps and detected by isoelectrofocusing microheterogeneity, proved to be the same for BASP1 samples isolated from both brain and non-nervous tissues. Therefore, in spite of different physiological consequences, biochemical functions of BASP1 must also be similar in different tissues.

Neuronal protein GAP-43 is a member of novel group of brain acid-soluble proteins (BASPs).

A group of brain acid-soluble proteins (BASPs) is preliminarily characterized. In some respects BASPs are similar to high mobility group (HMG) proteins, but in contrast to HMG, all BASPs are very acidic (pI 4.4-4.6) and show abnormal mobility during SDS-PAGE electrophoresis. BASP2-1 and BASP2-2 are identified as the two forms of neuronal protein GAP-43 (B-50, pp46, F1, neuromodulin). BASP1 and BASP3 are apparently novel brain proteins.

Tissue specificity of nucleo-cytoplasmic distribution of HMG1 and HMG2 proteins and their probable functions.

The levels and distribution between nucleus and cytoplasm of HMG1 and HMG2 proteins have been investigated in different tissues of mammals. In lymphoid tissues and testis high amounts of these proteins are present in both nuclei and cytoplasm, while in the hepatic tissues and brain they accumulate in cytoplasm, mainly in the cytosol. In particular, very low amounts, if any, of HMG1 and 2 are present in the nuclei active for DNA replication (rat regenerating liver and primary hepatoma) or transcription (adult liver and brain). Therefore, it appears that HMG1 and 2 are not necessary for these processes. On the other hand, nuclear (chromosomal) HMG1 and 2 are characteristic for the tissues containing undifferentiated cells: lymphoid tissues, testis, neonatal liver. These proteins are bound to the chromatin regions solubilized early by sonication or DNase action. Comparison of the data obtained for different tissues shows an inverse correlation between the amounts of chromosomal HMG1 and 2, on the one hand, and of histone H1(0), on the other hand. These results suggest that chromosomal HMG1 and 2 take part in the processes that occur during cell differentiation, while histone H1(0) is induced to preserve differentiated cells from dedifferentiation.

Thymidine uptake in bacteria: the effect of purine nucleosides.

The kinetics of thymidine uptake by Escherichia coli and Bacillus subtilis cells in the presence of adenine and guanine nucleosides was investigated. The initial concentration of thymidine in the growth medium was 0.35 microng/ml while the initial concentration of purine nucleosides ranged from 25 to 250 microng/ml. Adenine nucleosides when present at a concentration more than 50 microng/ml strongly inhibit thymidine uptake by the bacteria. The duration of the inhibition depends on the initial concentration of adenine nucleoside in the growth medium. At an initial concentration of deoxyadenosine (or adenosine) of 250 microng/ml the time of inhibition of thymidine uptake was about 60 min. During this period thymidine is almost completely preserved from the action of bacterial thymidine phosphorylase. Guanine nucleosides (guanosine or deoxyguanosine) do not markedly inhibit thymidine uptake by bacteria even at a concentration of 250 microng/ml. It is shown that they do protect thymidine from the phosphorolytic action of the thymidine phosphorylase although much less effectively than adenine nucleosides. It is suggested that some areas in the bacterial membrane where thymidine phosphorylase is located are not available to guanine nucleosides.

The absence of histone H1 from the chromatin fraction obtained by sonication of calf thymus nuclei under "quasiphysiological" ionic conditions.

The minor chromatin fraction was isolated from the sonicated calf thymus nuclei on the basis of its differential solubility in the "quasiphysiological" salt medium (0.1 M KCl-0.05 M NaCl-l mM MgCl2-1 mM CaCl2). Histone Hl is almost completely absent from this fraction. DNA isolated from this fraction occurs in three discrete low mol. wt. fragments. The fraction of chromatin which lacks histone Hl can also be obtained by two other methods. On of them consists in salt precipitation of the chromatin gel and its subsequent sonication. The second method includes precipitation of the sonicated chromatin gel by salts. In the first case the properties of the chromatin fraction which remains in the supernatant after centrifugation closely resemble those of the original salt-soluble nuclear fraction. The second method yields supernatant fraction also lacking histone Hl but containing heterogeneous DNA. Comparisons were also made of the sonically-solubilized nuclear fractions obtained in the complete salt medium and its mono and divalent cationic constituents.

Dependence of transfection efficiency of calcium treated Escherichia coli cells on bacterial genotype and form of Lambda DNA.

The transfecting activity of linear lambda DNA is 100 times higher in calcium treated E. coli K12 (lambda i434) than in non-lysogenic strains: the levels of transfection are 1-2.10(7) and 1-2.10(5) infective centers per 1 mug of lambda DNA respectively. The high efficiency of lysogenic cells transfection is not due to the spontaneously liberated "helper" phage. Evidently, it is called forth by transfecting DNA-prophage recombination or/and by inhibition of nuclease activity in lysogenic cells. Both ring forms lambda DNA (supercoiled and open circles) show very low infectivity, if any, in calcinated cells.