Kinetics and Regulation of Smac/DIABLO Release From Mitochondria of Cancer Cells Exposed to Apoptogenic Stimulus

Summary: The majority of currently applied anticancer drugs and radiotherapy activate mitochondrial pathway of apoptosis through DNA damage and inhibition of key enzymes involved in the survival signaling pathways of cancer cells. That triggers the cascade of reactions leading to the activation of proapoptotic proteins from Bcl-2 family such as: Bad, Bid, Bax and Bak, which are involved in formation of specific channels or coformation of megachannels (mPTP) in outer mitochondrial membrane. These channels are responsible for the release of apoptogenic proteins such as cytochrome c, Smac/DIABLO, AIF, Omi/HtrA2, procaspase-9 and -3 and endonuclease G from mitochondrial intermembrane space. Smac/DIABLO (second mitochondria-derived activator of caspases/direct IAP binding protein with low pI) is second (after cytochrome c) activator of caspases, functioning through the release of caspases from inhibitory effects of IAPs (inhibitors of apoptotic proteases). Precursor of Smac/DIABLO is synthesized as a 239-amino acid protein. After reaching mitochondria the Smac/DIABLO precursor loses the N-terminal mitochondrial-targeting sequence and becomes the mature 21 kDa protein with apoptotic activity. It was shown, that Smac/DIABLO exists in two forms in the cell: inactive cytosolic short form (Smac/DIABLO-S) and active mitochondrial long form (Smac/DIABLO-L). Because differences between both forms occur only at N-terminus, probably the N-terminal residues of Smac/DIABLO are responsible for its biological activity. Inhibitory function of Smac/DIABLO on IAPs depends on competitive blocking of BIR3 domain, which inhibits binding of caspase-9. Apart from this, changes occur at spatial structure of BIR2 linker, thus inhibiting IAPs interaction with caspase-3 and-7. Smac/DIABLO is released from mitochondria not only in mitochondrial, but also in death receptor pathways of apoptosis. It results from the fact, that apoptotic signal derived from death receptors through caspase-8 is too weak for the complete activation of executory caspases (-3, -6 and -7) in cancer cells. The death receptor signal must be amplified through the release of Smac/DIABLO from mitochondria and removal of inhibitory influence of IAPs on caspases. It is interesting, that during this event cytochrome c is not released from mitochondria, which may indicate a different molecular mechanism of release of both proteins. In our recent study homeostatic confocal microscopy was used for precise evaluation of Smac/DIABLO kinetics in cancer cells stimulated to apoptosis. These studies revealed the release of Smac/DIABLO-GFP from mitochondria beginning immediately after cancer cell exposure to cytotoxic drug. Inhibition of m-calpain (regarded as a Bax activator) delayed the escape of Smac/DIABLO-GFP from mitochondria and inhibited apoptosis. Our data based on laser scanning cytometry, confocal microscopy and immunoelectron microscopy as well as literature data indicate the key role of Bax and other proteins implicated in formation of megachannels in outer mitochondrial membrane in the regulation of Smac/DIABLO release from mitochondrial intermembrane space.

Key words: apoptosis anticancer drugs, cytochrome c, Smac/DIABLO, Bad, Bid, Bax and Bak mitochondria, caspases, cancer cells laser scanning cytometry, confocal microscopy and immunoelectron microscopy.
 

Transcriptional Regulation of Adipogenesis

Summary: The major role of adipocytes is storing triacylglicerol during periods of energy excess and its mobilization in times of energy deprivation. These cells act also as a secretory organ, that synthesize many paracrine and endocrine hormones, among others leptin. Adipogenesis is a cascade of molecular events that leeds to conversion of mesenchymal cells into preadipocytes and finally into mature adipocytes. This process is controlled by several transcription factors, including predominantly members of C/EBP and PPAR families. Polymorphism of genes belonging to C/EBP and PPAR families may have an influence on human obesity and fatness traits in livestock species.

Key words: adipogenesis, transcription factors, C/EBP, PPAR, CREB, fat
 

Gene Therapy of Hematopoietic Cells: Development of Strategies and Vectors.

Summary: Last decade witness development of gene therapy strategies in hematopoietic system from attempts of correcting mutated genes in monogenic diseases till research on applications in transplantology, cancer therapy, and AIDS. Intensive research concerns therapeutic use of genetically modified human stem cells with encouraging results achieved in animal models. Fulfillment of gene therapy potential requires improvements in gene transfer methods and providing their safety. All gene therapy vectors, both viral-based and biochemical, are characterized by certain limitations and safety risks. Upgrading of viral and biochemical vectors are governed by the same requirements for transgene delivery through the cell membrane barrier, escape from endosomal compartment, and finally entry to the nucleus and achieving controlled gene expression at therapeutic level. Despite significant methodological progress, providing safety of gene therapy still represents a challenge and requires further intensive research.

Key words:  gene therapy, hematopoietic cells, viral vectors, nonviral vectors
 

The COP9 Sygnalosome and its Role in Regulation Protein Stability in Eukaryotes

Summary: The COP9 signalosome (CONSTITUTIVE PHOTOMORPHOGENIC 9) (called often CSN) is a multiprotein, nuclear complex existing commonly in Eucariotes. Numerous studies have shown that the CSN is engaged in many processes such as photomorhogenesis, cell cycle control and differentiation. The CSN is implicated in ubiquitin mediated protein degradation and recently have been shown to interact with three ubiquitin ligases: SCF (SKP1, Cullin/CDC53, F-BOX), MDM2 (MURINE DUBLE MINUTE 2), COP1 (CONSTITUTIVE PHOTOMORPHOGENIC 1). Two activities associated with the CSN have been identified so far: a protein kinase and NEDD8 (NEURAL PRECURSOR CELL-EXPRESSED DEVELOPMENTALLY DOWN-REGULATED 8) deconjugation (deneddylation) that may determine stability of CSN interacting proteins. Additionally, the CSN appears to be involved in cellular transport of ubiquitinated proteins from the nucleus into the cytosol and triggers their degradation by the 26S proteasome. The CSN is built by 8 subunits. Two of them contain the MPN (Mpr1, Pad1p, N-end), domain, the rest contain PCI domain. PCI (Proteasom, Cop9, eIF3) domain is a characteristic element of two another multiprotein complexes: 26S proteasome and eIF3. These facts suggest that these three complexes may be involved in multiple control of protein stability.

Key words:  COP9 signalosome, ubiquitin ligases, protein degradation
 

Influence of Lipids on DNA Polymerases Activity

Summary: Lipids are very important structural and functional elements of cell membranes system. Being component of cytoplasm and nucleus regulate intracellular processes. Suppression of isolated and purfied DNA related polymerases activity by fatty acids, complex lipids and lipid peroxidation products observed in vitro can be a part of mechanisms controlling cell proliferation and differentiation in vivo.

Key words: polymerases, fatty acids, complex lipids, lipid peroxidation products
 

CD34 Antigen and CD34-positive Cells

Summary:  The hematopoietic stem cell, the common ancestor of all types of blood cells, is one the best-characterized stem cells in the body. It is suggested that stem cells derived from bone marrow are capable of giving rise to multiple cell types. These include neural cells, skeletal muscle, cardiac muscle, and hepatic cells, as well as epithelia of the gut, skin, lung and kidney. The antigen CD34 is the major positive marker for human hematopoietic stem, and progenitor cells. Chemically, CD34 is sialomucin – transmembrane glycoprotein. There are two species of CD34, full-length and truncated, derived from an alternative splicing mechanism of CD34 mRNA. It is suggests that the CD34 plays a role in adhesion to the stromal microenvironment of bone marrow. CD34 is expressed by all small-vessel endothelial cells. Expression of CD34 mRNA has also been detected in liver, spleen, and thymus. Contrary to current dogma, CD34 is expressed by mature  mast cells. In addition, there are CD34+ cells called fibrocytes that are present in monocyte fractions of human blood. In this paper is presented the current  view on the recent research  results of CD34 molecule  and CD34 positive cells.

Key words:  CD34 antigen, structure, function, CD34-positive cells, stem cells.

Micronuclei in Human Cells – Formation, Contents, Diagnostic and Prognostic Application

Summary: Micronuclei are formed during mitotic cell division and they incidence in cytoplast of daughter cell near the nucleus. The two basic phenomena leading to the formation of micronuclei are chromosome breakage and disfunction of the mitotic apparatus. Micronuclei can contain whole chromosomes or chromatids, acentric fragments or chromatid fragments. Several studies have shown that micronuclei frequencies and their contents is changing and depents on diseases, aging process and different environmental factors. Analisis of micronuclei frequencies and their contents is used to measure chromosome damage at the level of single cell and to assess susceptibility to degenerative and malignant diseases and to assess degree of exposure to environmental factors.

Key words:  micronuclei, cytochalasin B, binucleated cells, chromosomal aberrations, genetic instability
 

Nature and Functions of Cajal Bodies in the Light of New Research

Summary: Cajal bodies were first described 100 years ago in mammalian neurons. At the present moment, it is known that CB’s are highly conserved structures found in somatic and generative cells of plants and animals. CB’s marker protein is coilin, a phosphoprotein which is most probably responsible for location of many macromolecules in Cajal bodies. The presence of small nuclear RNAs (snRNAs) in CB is the evidence that this structure takes part in spatial organization of elements participate in pre-mRNA splicing. Additionally, the association with genes that code snRNA as well as the presence of their transcription and processing factors in CB strongly suggest that Cajal bodies participate in biogenesis of small nuclear RNAs (snRNAs). Furthermore there was shown the spatial and functional relationship of CB with nucleoli. Probably Cajal bodies also participate in some steps of  histone and small nucleolar RNAs (snoRNAs) genes expression.

Keywords: Cajal body (CB), snRNP, pre-mRNA splicing.
 

Heat Shock Proteins – Molecular Perpetuum Mobile

Summary:Heat Shock Proteins [HSP, molecular chaperones] are among the most highly conserved protein in the biosphere and play very important role in eucariotic and procariottic cells, providing them with protection from environmental factors and guaranting a steady state of cell metabolism. HSPs are defined as a group of unrelated proteins that mediate the correct folding, assembly, reparation, translocation across membranes and degradation of other proteins and simultaneously are not their functional components. HSPs can be divided into six subfamilies according to their molecular weights: the large Hsp100 family, intermediate HSP90, 70, 60 ,40 families and the small HSPs of 18-30 kDa. Expression of most HSPs is induced by temperature and other molecular stressors including nutrient deprivation, oxygen radicals, metabolic deterioration, viral and bacterial infections, cytokines and UV irradiation. This article presents general characteristics of HSPs and their role in modulating both inflammatory and immune response, including possible connection with many diseases.

Key words:  heat shock proteins, immunity, apoptosis
 

Genomic Imprinting in Plants

Summary: The epigenetic phenomenon of genomic imprinting occurs among both plants (flowering plants) and animals (mammals) reflecting as parent-of-origin effects on the expression of imprinted genes in offspring. Imprinting results in non equivalent expression of maternally and paternally derived alleles in an individual. It means that one of the alleles is supressed during development in sex-specific manner. This reviews focuses on imprinting in plants. Embryo and endosperm in flowering plants are the result of double fertilization, a special event characteristic for this group of plants only. Imprinting in plants affects the endosperm development but not the embryo development in contrast to mammals where the consequence of imprinting is inviability of embryos with uniparental genome (male or female) contribution (androgenic embryos, parthenogenetic/gynogenetic embryos, respectively). There is molecular and genetic evidence for so-called maternal-effect genes expressed in both diploid sporophyte and in haploid female gametophyte. Several such genes involved in seed development were described in Arabidopsis and in Zea. Most interest were placed on the group of Arabidopsis genes MEDEA (MEA), FERTILIZATION-INDEPENDENT SEED2 (FIS2) and FERTILIZATION-INDEPENDENT ENDOSPERM (FIE) responsible for the supression of an egg cell and central cell nucleus divisions before fertilization. Analysis of early embryogenesis in Arabidopsis clearly indicated that  the expression of paternal alleles is delayed during seed development being an effect of genomic imprinting. It is thus obvious that in the absence of paternally inhered alleles, early divisions of zygote are programmed by maternal copies. Differential expression of paternal and maternal alleles was stated for 20 genes in Arabidopsis expressed in early seed development. Mechanism of imprinting in plants are considered to be cytosine methylation and chromatine condensation similarly as is postulated in animals but also paramutation and transgene silencing could be involved in imprinting in plants. The role of imprinting in plant reproduction specially in apomixis is also presented.

Key words: plants, genomic imprinting, DNA methylation, Polycomb proteins, embryo, endosperm, apomixis
 

Oligo- and Monomeric Ubiquitin Ligases E3 with Ring Finger Domain - Structure and Function

Summary: In the article the basic information is given about proteolysis carried out with participation of ubiquitin/proteasome 26S system, the highly regulated process indispensable for cell functioning. The main subject of the article is the description of proteins containing the RING finger domain and functioning as ubiquitin ligases E3 involved in the ubiquitination of proteins destined to the degradation in the proteasome. The importance of RING finger domain in functioning of oligomeric (SCF, Parkin, VCB, APC/C) as well as monomeric (c-Cbl, MDM2, BRCA1, CHFR, IAP) ubiquitin ligases was stressed. Moreover it was pointed out, that the abnormalities of expressions and functioning of the ubiquitin ligases may be connected with various diseases.

Key words:  proteolysis, ubiquitin, proteasome 26S, ubiquitin ligases E3, RING finger domain