Andrei A. Karelin

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.

Generation of peptides by human erythrocytes: facts and artifacts.

Previously reported data on peptide composition of human erythrocyte lysate were obtained under conditions that did not exclude proteolytic degradation of hemoglobin in the process of peptide isolation. Comparative chromatographic analysis of the diluted erythrocyte lysate incubated in acidic conditions with or without proteolytic enzyme inhibitors showed that several peptides earlier identified as intraerythrocyte ones in fact result from hemoglobin degradation by erythrocyte acidic protease(s) during incubation of the lysate. A rational scheme excluding postlysis proteolysis was developed for isolation of peptide fraction. Further analysis resulted in determination of structure and content of about 50 endogenous intraerythrocyte hemoglobin fragments. A primary endopeptidase splitting of α‐ and β‐globin chains followed by consecutive exopeptidase trimming of primary fragments is suggested as a degradation mechanism. The intraerythrocyte peptides were shown to differ from peptides excreted by the erythrocytes to the extracellular medium in the primary culture. It was also found that intraerythrocyte peptides cannot play the role of precursors of hemoglobin fragments present in tissue extracts.

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.

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.

Peptides from bovine brain: structure and biological role.

Fractionation of bovine brain extracts followed by automatic Edman sequencing of individual components resulted in identification of 107 endogenous peptides formed from functional proteins (haemoglobin, myelin basic protein, cytochromecoxidase, etc) or unknown precursors. Several of the newly identified brain peptides demonstrate different types of biological activity; some of the substances show considerable overlap with the known biologically active peptides. It is suggested that these peptides should participate in regulation of extracellular and intracellular biochemical processes. A concept of ‘tissue‒specific peptide pool’ is formulated describing a novel system of peptidergic regulation, complementary to the conventional hormonal and neuromodulatory systems. According to that description functional proteins provide their proteolytically derived fragments for maintaining the tissue homeostasis by modulating the availability of peptide receptors to respective ‘true’ ligands.

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.

Endogenous fragment of hemoglobin, neokyotorphin, as cell growth factor.

It is shown that neokyotorphin (the alpha-globin fragment 137-141) stimulates proliferation of normal cells (murine embryonic fibroblasts, red bone marrow and spleen cells) and tumor cells (murine melanoma and transformed fibroblasts L929) in the absence or in the presence of fetal bovine serum. In contrast to serum deprivation conditions, the ability to potentiate L929 cell growth in the presence of fetal serum is strongly cell density dependent. The peptide also enhances the viability of L929 cells, murine embryonic fibroblasts and of the primary cultures of murine red bone marrow cells and splenocytes under serum-deprivation conditions for at least 72 h. The results of flow cytometry analysis suggest that the effect of neokyotorphin on survival of L929 cells in serum-free culture medium is due to maintenance of cell proliferation in the absence of growth factors. Along with cell cycle progression the peptide induces reversible reduction of L929 cell size.

Proliferative Activity Of Neokyotorphinrelated Hemoglobin Fragments In Cell Cultures

alpha-Hemoglobin fragments alpha-(133-141), alpha-(134-141), alpha-(135-141), alpha-(137-141), alpha-(134-140), alpha-(133-138), alpha-(134-140) and alpha-(137-138) stimulate L929 tumor cell proliferation, alpha-(134-141) being the most active. alpha-(134-141) stimulates proliferation of M3 melanoma cells, murine embryonic fibroblasts, primary cultures of red bone marrow and spleen cells. In L929 cells the effect of alpha-(134-141) is cell density independent; in M3 cells alpha-(137-141) and alpha-(134-141) are most active at density 10,000 cells/well (96 well plate) independently on FBS content.