Elena Yu. Blishchenko

A novel system of peptidergic regulation

Systematic analysis of structure and biological activity of peptide components of tissue extracts and biological fluids allows us to formulate a novel concept of a peptidergic regulatory system, complementary to the conventional regulatory systems (i.e. nervous, endocrine and paracrine systems). According to that concept, the proteolytic degradation of tissue proteins carried out by a specific and regulated system of tissue‐specific enzymes and protein substrates gives rise to a large group of peptides, which we define as tissue‐specific peptide pool. As a result, functional proteins provide their proteolytically derived fragments for maintaining tissue homeostasis.

Family of hemorphins: co-relations between amino acid sequences and effects in cell cultures.

Hemorphins, i.e. endogenous fragments of beta-globin chain segment (32-41) LVVYPWTQRY(F) suppress the growth of transformed murine fibroblasts L929 cell culture, the effect is due to cytotoxicity and inhibition of cell proliferation. The contribution of cytotoxicity depends on the presence of Leu(32): VV-hemorphins, except VV-hemorphin-4, exhibit cytotoxicity significantly higher than respective LVV-hemorphins. Decrease of cell number induced by hemorphins depend on the extent of N- and C-terminal degradation of hemorphins: VV-hemorphins in most cases are more active than LVV-, V-hemorphins, and hemorphins. In the group of VV-hemorphins the activity of VV-hemorphin-5 (valorphin) is significantly higher than of VV-hemorphin-7, VV-hemorphin-6, and VV-hemorphin-4, meaning that the presence of C-terminal Gln is important for suppressing of cell number. The amino acid sequence VVYPWTQ corresponding to valorphin was identified as important for manifestation of the both cytotoxic and antiproliferative effects.

LVV‐ and VV‐hemorphins: comparative levels in rat tissues

Screening of hemorphins in extracts of rat lung, brain, heart and spleen was carried out. The threshold for detection of hemorphins was 0.01 nmol for spleen and 0.05 nmol for other tissues. Both the content and the composition of hemorphins differed significantly in the tissues analyzed. Heart and lung extracts were rich in these peptides, the content of the most abundant components reaching 16–44 nmol/g of tissue. In contrast, spleen and brain contained much lower amounts of hemorphins, i.e. about 0.3–2.6 nmol/g of tissue. The most represented hemorphin in lung, heart and brain was VV‐hemorphin‐5, while the content of other members of the hemorphin family depended significantly on the tissue analyzed: lung extract was also rich in LVV‐hemorphin‐5, heart contained similar amounts of LVV‐hemorphin‐7 and LVV‐hemorphin‐5 and brain of LVV‐hemorphin‐6. In contrast, the hemorphin family in spleen was represented mainly by C‐terminally shortened VV‐hemorphins, i.e. VV‐hemorphin‐4 and VV‐hemorphin‐3. The levels of hemorphins in all cases were sufficient to activate the opioid receptors of the respective tissues.

Antitumor effect of valorphin in vitro and in vivo: combined action with cytostatic drugs.

The action of the cytostatic drugs (epirubicin and vincristine) in combination with the endogenous antiproliferative £]-hemoglobin fragment (33-39), valorphin, was studied in tumor (L929 and A549) cell cultures, primary culture of murine bone marrow cells and in murine model of breast carcinoma in vivo. Simultaneous application of 1 µM valorphin and 1 µM epirubicin, in vitro, did not result in an additive suppressive effect on cell culture growth. Additive effects were achieved with alternating applications of the peptide and the drugs, namely, 0.5 µM (but not 1 µM) epirubicin added 24 h prior to 1 µM valorphin; 1 µM valorphin added 48 h prior to 0.1 µM epirubicin, or 0.1 µM vincristine, or 0.05 µM vincristine, which resulted in 100% cell death in the both series with vincristine and up to 78% cell biomass reduction in the experiments with epirubicin. In the in vivo model (female BLRB mice with subcutaneously inoculated syngeneic mammary carcinoma), simultaneous treatment with 25 mg/m2 epirubicin and 1 mg/kg valorphin resulted in 42% of tumor growth inhibition, as compared with the negative control group and 22% inhibition as compared with the epirubcin-treated group (at 20th day of treatment). Survival was significantly improved (69% compared to 39% in the group treated with epirubicin only) at day 26 after the treatment beginning.

Tumor Cell Cytolysis Mediated by Valorphin, an Opioid-Like Fragment of Hemoglobin β-Chain

Valorphin, an endogenous opioid-like hemoglobin fragment, is cytotoxic for L929 and K562 tumor cells in 10(-7)-10(-13) M concentration range. Because cytolytic effects induced by valorphin in K562 cells are inhibited by naloxone, opioid receptors should be involved in induction of valorphin-mediated tumor cell death. Three distinct cytolytic processes, differing in the onset time and the development time, take place with K562 cells within 10-18 h of incubation with valorphin. All three processes are not associated with apoptotic mechanism of cell death.

Neokyotorphin and neokyotorphin (1–4): secretion by erythrocytes and regulation of tumor cell growth

Human erythrocytes release neokyotorphin, the 137–141 fragment of hemoglobin α‐chain into the supernatant of red blood cells primary culture. However, the neokyotorphin fragment 1–4 that is formed together with neokyotorphin inside the red blood cells and in various tissues is not found in the supernatant. Both neokyotorphin and its 1–4 fragment were shown to stimulate proliferation of L929 tumor cells.

Stimulation of fibroblast proliferation by neokyotorphin requires Ca2+ influx and activation of PKA, CaMK II and MAPK/ERK

Neokyotorphin [TSKYR, hemoglobin α‐chain fragment (137–141)] has previously been shown to enhance fibroblast proliferation, its effect depending on cell density and serum level. Here we show the dependence of the effect of neokyotorphin on cell type and its correlation with the effect of protein kinase A (PKA) activator 8‐Br‐cAMP, but not the PKC activator 4β‐phorbol 12‐myristate, 13‐acetate (PMA). In L929 fibroblasts, the proliferative effect of neokyotorphin was suppressed by the Ca2+L‐type channel inhibitors verapamil or nifedipine, the intracellular Ca2+ chelator 1,2‐bis(2‐aminophenoxy)ethane‐N,N,N′,N′‐tetraacetic acid acetoxymethyl ester, kinase inhibitors H‐89 (PKA), KN‐62 (Ca2+/calmodulin‐dependent kinase II) and PD98059 (mitogen‐activated protein kinase). The proliferative effect of 8‐Br‐cAMP was also suppressed by KN‐62 and PD98059. PKC suppression (downregulation with PMA or inhibition with bisindolylmaleimide XI) did not affect neokyotorphin action. The results obtained point to a cAMP‐like action for neokyotorphin.

Met-enkephalin induces cytolytic processes of apoptotic type in K562 human erythroid leukemia cells

Cytolytic activity of Met‐enkephalin, an endogenous opioid peptide, was studied within the 10−7−10−17 M concentration range in K562 human erythroid leukemia cells. Cytolytic activity was determined by the trypan blue inclusion method after 13, 15 and 18 h of Met‐enkephalin co‐incubation with target cells. Discrete maxima of cytolytic activity were detected at concentrations of 10−9−10−10, 10−13 and 10−15 M. Cytolysis was accompanied by internucleosomal DNA fragmentation which is indicative of the mechanism of cell death being apoptosis.

Peptides comprising the bulk of rat brain extracts: isolation, amino acid sequences and biological activity

Chromatographic separation of rat brain extracts followed by automatic Edman sequencing of the major individual components resulted in identification of 61 endogenous peptides derived from known functional proteins (hemoglobin, myelin basic protein, cytochrome‐c oxidase, etc.) or unknown precursors. The results are compared with the data obtained earlier for bovine brain. Although the sequences of bovine and rat hemoglobin contain about 20% of amino acid substitutions, the families of structurally related peptides are very similar in both extracts. Several other proteins also give rise to identical or closely related peptide fragments in the two mammalian species. The outlined similarity extends almost exclusively to the most abundant peptides present in the extracts. The minor components show less overlap. Four hemoglobin‐derived peptides isolated from rat brain were shown to be biologically active in tumor cells. Eleven are identical to bioactive peptides from other species. Ten structurally overlap with bioactive peptides from other sources. The data obtained show similar biosynthetic pathways of pool components in different species, the resultant peptides being aimed at fulfilling related functions. Copyright

Fragments of functional proteins: role in endocrine regulation.

Systematic analysis of structures, localization, formation and biological activities of endogenous peptides derived from functional proteins, such as hemoglobin, myelin basic protein, immunoglobulins, etc., allowed establishing the basic features of that group of compounds. The sets of these peptides in mammalian tissues, or “tissue-specific peptide pools” are: (i) tissue specific; (ii) stable at normal conditions; (iii) conservative in the same tissues of different mammalian species; (iv) dependent on the general state of homeostasis of tissue or the whole organism. Formation of such peptides has features of both conformation and site specificity and also involves the action of carboxy- and amino-peptidases. As a result, the families of structurally related families of peptides are generated. The fragments of functional proteins exhibit a wide range of the biological effects, characteristic both for hormones and parahormones, from hormone-releasing to growth-regulatory activity. At the same time, the molecular mechanisms of action of the majority of such peptides are unknown. On the basis of the data obtained the components of tissue-specific peptide pools are considered to form a novel regulatory system, complementary to other peptidergic systems such as hormonal, nervous, immune, etc. The biological role of the fragments of functional proteins in vivo and the patterns of interaction with other regulatory systems are suggested.