PEDF - A Noninhibitory Serpin with Neurotrophic Activity.

The pigment epithelium-derived factor (PEDF) is a glycoprotein with a molecular weight of 50 kDa belonging to the noninhibitory serpin family. It regulates several physiological processes, such as stimulation of retinoblastoma cell differentiation into neuron cells, and facilitation of the growth and viability of photoreceptor cells and neurons of the central nervous system. Moreover, this factor protects neuronal cells against apoptosis. PEDF is not only a neurotrophic factor, but also a natural angiogenesis inhibitor. This protein, as well as its biologically active fragments, possesses significant neuroprotective, neurotrophic, and antiangiogenic capabilities. The precise molecular mechanisms underpinning the effects of PEDF are still not quite clear. However, this protein generates great interest as a promising drug for the therapy of a wide range of neurodegenerative, ophthalmological, and oncological diseases. This review is a summary of what is known today about the structural features, biochemical properties, and multimodal functions of PEDF.

Antitumor and immunomodulating effects of a fragment of HL-60 cell differentiation factor in mice with lewis pulmonary carcinoma.

A six-member HLDF6 peptide, fragment of HL-60 cell differentiation factor, exhibited antimetastatic and immunomodulating effects.

Granulocyte differentiation inducer, hexapeptide HLDF-6, decreases cytotoxic effect of tumor necrosis factor on HL-60 cell line.

The effect of hexapeptide HLDF-6, the granulocytic differentiation inducer, on the tumor necrosis factor alpha (TNF-alpha)-induced differentiation and apoptosis of human promyelocytic leukemia HL-60 cells has been investigated. Costimulation of HL-60 cells with HLDF-6 and TNF-alpha enhanced granulocyte differentiation, whereas the level of monocyte differentiation remained unchanged; however, the cytotoxic action of TNF-alpha on these cells decreased. The protective effect of HLDF-6 peptide did not depend on activation of NF-kappaB (nuclear transcription factor). Since HLDF-6 peptide decreases the number of cells entering apoptosis caused by C(2)-ceramide, a mediator of TNF-induced apoptosis, and also reduces TNF-alpha-mediated activation of caspase-3, we have proposed the hypothesis that HLDF-6 increases resistance of HL-60 cells to the TNF-alpha cytotoxic effect due to inhibition of some stages of mitochondria-dependent apoptotic signaling.

Hexapeptides HLDF-6 and PEDF-6 restore memory in rats after chronic intracerebroventricular treatment with beta-amyloid peptide Abeta(25-35).

Effects of homologous peptides HLDF-6 and PEDF-6 on behavior of animals with experimental Alzheimer's disease induced by chronic intracerebroventricular administration of beta-amyloid peptide Abeta(25-35) were studied in the zoosocial recognition test and Morris water maze. Peptides HLDF-6 and PEDF-6 possessed neuroprotective activity and counteracted the toxic effect of Abeta(25-35). Peptides HLDF-6 and PEDF-6 mainly improved long-term memory and working memory, respectively.

Experimental model of hemorrhagic stroke: rabbit immunization with HL-60 promyelocytic cell differentiation factor.

Rabbits immunized with synthetic HLDF differentiation factor developed hemorrhagic stroke with thrombosis of small cerebral vessels and destruction of vascular endotheliocytes. The severity of stroke correlated with serum level of antibodies to differentiation factor. The role of different sites of HLDF molecule in the induction of clinical signs of hemorrhagic stroke was studied.

Intermediate amyloid oligomers of lysozyme: Is their cytotoxicity a particular case or general rule for amyloid?

In the current study we investigated the molecular mechanisms of cytotoxicity of amyloid oligomers of horse milk lysozyme. We have shown that lysozyme forms soluble amyloid oligomers and protofibrils during incubation at pH 2.0 and 4.5 and 57 degrees C. These structures bind the amyloid-specific dyes thioflavin T and Congo Red, and their morphology and size were analyzed by atomic force microscopy. Monomeric lysozyme and its fibrils did not affect the viability of three cell types used in our experiments including primary murine neurons and fibroblasts, as well as neuroblastoma cell line IMR-32. However, soluble amyloid oligomers of lysozyme caused death of all these cell types, as estimated by flow-cytometry counting dead cells stained with ethidium bromide. The primary cell cultures appeared to be more sensitive to amyloid than neuroblastoma cell line IMR-32. Amyloid cytotoxicity depends on the size of oligomeric particles: samples containing 20-mers formed at pH 4.5 were more toxic than tetramers and octamers present in the solution at pH 2.0. Soluble amyloid oligomers can self-assemble into doughnut-like structures; however, no correlation was observed between the amount of the doughnut-like structures in the sample and its cytotoxicity. The fact that the intermediate oligomers of such an abundant protein as lysozyme display cytotoxicity confirms a hypothesis that cytotoxicity is a common feature of protein amyloid. Inhibition of intermediate oligomer formation is crucial in preventing amyloid pathogeneses.

Amyloidogenic properties of the artificial protein albebetin and its biologically active derivatives. The role of electrostatic interactions in fibril formation.

The artificial protein albebetin (ABB) and its derivatives containing biologically active fragments of natural proteins form fibrils at physiological pH. The amyloid nature of the fibrils was confirmed by far UV circular dichroism spectra indicating for rich beta-structure, thioflavin T binding assays, and examination of the obtained polymers by atomic force microscopy. Fusing of short peptides--octapeptide of human alpha(2)-interferon (130-137) or hexapeptide HLDF-6 (41-46) of human leukemia differentiation factor--with the N-terminus of ABB led to increased amyloidogenicity of the protein: the rate of fibril formation increased and the morphology of fibrils became more complex. The presence of free hexapeptide HLDF-6 in the ABB solution had the same effect. Increasing ionic strength also activated the process of amyloid formation, but to less extent than did the peptides fused with ABB or added to the ABB solution. We suggest an important role of electrostatic interactions in formation of ABB fibrils. The foregoing ways (addition of salt or peptides) allow decrease in electrostatic repulsion between ABB molecules carrying large negative charge (-12) at neutral pH, thus promoting fibril formation.

Hemodynamic effects of peptide fragments of differentiation factor HLDF.

Hemodynamic activity of peptides from differentiation factor HLDF (promyelocytic HL-60 line) was studied on WKY and SHR-SP rats. Intravenous infusion of the test peptides was accompanied by changes in blood pressure and heart rate, which depended on the structure of peptides and functional activity of the organism and differed in normotensive and hypertensive animals.

HLDF-6 peptide affects behavioral reactions and organism functions dependent on androgen hormones in normal and castrated male mice.

The hexapeptide Thr-Gly-Glu-Asn-His-Arg (HLDF-6), which was first identified as an active fragment of the human leukemia differentiation factor (HLDF) molecule, displays differentiation-inducing, neuroprotective and anti-drug abuse activities. Most of its in vivo effects were revealed only on male animals. We have studied HLDF-6 effects on a variety of organism functions and behavioral reactions, which are known to be dependent on androgen steroid hormones, both on castrated and normal (sham-operated) animals. Male NMRI mice were castrated or sham-operated at the age of 55 days (after puberty). After that, HLDF-6 peptide was injected daily during 3 weeks, followed by behavioral, morphological and biochemical testing. HLDF-6 increased testosterone level (1.5- to 2-fold) both in sham-operated and castrated animals. Sexual activity and pain sensitivity, which are strongly reduced in castrates, were completely or partially recovered by HLDF-6. At the same time, the peptide caused some effects similar to castration in sham-operated animals: aggression and locomotor activity were decreased; oral grooming was prolonged. Morphological studies of accessory sex glands showed that HLDF-6 partially normalizes the morphology and functional activity of seminal vesicles in castrates, but it does not prevent castration-induced apoptosis of prostate epithelial cells. Based on these observations, we can assume that HLDF-6 peptide displays at least two effects on androgen hormones metabolism in males: it stimulates testosterone biosynthesis by both testes and adrenals and simultaneously inhibits its conversion to dihydrotestosterone (DHT), most probably by diminution of 5alpha-reductase isoform 1 mRNA expression.

Different mechanisms of protective and differentiative activities of homological peptides TGENHR and TQVEHR.

Previously we identified a six-membered fragment 354TQVEHR359 of the C-terminal part of the PEDF (Pigment Epithelium-Derived Factor) differentiation factor molecule that shares homology with fragment 41TGENHR46 of the HLDF (Human Leukemia Differentiation Factor) differentiation factor molecule, which is responsible for its differentiation activity. HLDF has been isolated from the culture medium of human promyelocytic leukemia cell line HL-60. Hexapeptides HLDF-6 (TGENHR) and PEDF-6 (TQVEHR) corresponding to these HLDF and PEDF molecule fragments, which were previously shown to induce cell differentiation (Kostanyan et al. (2000) Russian Journal of Bioorganic Chemistry, 26, 505-511), also have neuroprotective properties. Both peptides prevent degeneration of Purkinje cells of rat cerebellar vermis upon chemical hypoxia induced by sodium azide in vivo; this effect is also observed on a behavioral level. Peptide HLDF-6 but not PEDF-6 promotes survival of HL-60 cells upon chemical hypoxia. Peptides HLDF-6 and PEDF-6 affect different second messenger biosynthesis systems in HL-60 cells. HLDF-6 diminishes cyclic AMP level in those cells due to adenylate cyclase inhibition, while PEDF-6 inhibits phosphatidylinositol-specific phospholipase C stimulated by aluminum tetrafluoride anions.

HLDF-6 peptide relieves symptoms of abstinence syndrome during experimental opium abuse.

Experiments were performed on rats with opium abuse induced by chronic administration of morphine in increasing doses. We studied the effect of HLDF-6 peptide on symptoms of naloxone abstinence. Repeated administration of HLDF-6 peptide in a dose of 0.2 mg/kg 24 and 0.5 h before naloxone relieved the major symptoms of the abstinence syndrome. A possible neurochemical mechanism underlying the effect of HLDF-6 peptide is inhibition of enkephalinase A in structures of the endogenous antinociceptive system.

Autoimmune responses to amyloid structures of Abeta(25-35) peptide and human lysozyme in the serum of patients with progressive Alzheimer's disease.

We have found an increased level of serum antibodies to the prefibrillar structures of both Abeta(25-35) peptide and human lysozyme in Alzheimer's disease (AD) patients compared to age-matched controls, indicating that autoimmunity is implicated in AD. In the serum of AD patients with a long-term duration (>15 years) the titer of serum antibodies to aggregates of Abeta(25-35) peptide increased by approximately 5-fold, whilst the antibody titer to lysozyme protofilaments decreased by approximately 8-fold compared to patients with AD duration of <5 years. The content of immunoglobulins of the A, G and M types declined, particularly in AD duration of >15 years. An increase in the concentration of immune complexes and higher lysozyme activity was detected in the serum of all patients and this was suggestive of an inflammatory reaction. We propose that the autoimmune response to different amyloid structures in AD can be viewed as a clearance pathway targeting amyloid development. Autoimmune response can be exploited as a marker of ongoing protein aggregation and hence be used as a diagnostic feature of AD.

Use of HLDF-6 peptide for correction of disturbances in the endogenous opioid system in offspring of morphine-tolerant animals.

We studied the effect of bioactive peptide HLDF-6 on functional activity of the endogenous antinociceptive system in the offspring of morphine-tolerant animals. Disturbances in this system included changes in the thermonociceptive threshold and enkephalinase A activity in various brain structures. The peptide acted as a potent regulator of the homeostasis in systems responsible for the synthesis and catabolism of endogenous opioids. HLDF-6 effectively corrected disorders of the endogenous antinociceptive system.

Soluble Fas antigen in the serum of patients with colon cancer.

Serum concentration of soluble Fas antigen (sFas) was measured in 60 normal subjects and 33 patients with colon cancer. The incidence of sFas detection and its serum content were higher in patients with colon cancer compared to normal subjects. No relationships between the incidence and level of sFas and patient's sex, age, duration and stage of the disease were found. Serum content of sFas tended to increase in patients with metastases to regional lymph nodes, liver, and lungs. The role of sFas as a marker predicting clinical course and outcome of colon cancer is discussed.

Leptin and apoptosis inhibitor soluble Fas antigen in the serum of patients with osteosarcoma and neuroectodermal bone tumors.

Serum contents of leptin and soluble Fas antigen were measured in 28 patients with osteosarcoma, 7 with neuroectodermal bone tumors, and 17 healthy subjects. The incidence and levels of soluble Fas antigen in patients with osteosarcoma and neuroectodermal bone tumors were higher than in healthy subjects and did not depend on sex and age in both healthy subjects and patients. Serum concentration of leptin in women was higher than in men (both in healthy controls and patients) and was lower in healthy subjects compared to patients with osteosarcoma and neuroectodermal bone tumors. A trend to a negative correlation between the concentrations of leptin and soluble Fas antigen in female patients with osteosarcoma and male patients with neuroectodermal bone tumors was revealed. The role of leptin and soluble Fas antigen in the pathogenesis of primary bone tumors is discussed.