Gelina S. Kopeina

Lipid-protein nanodiscs for cell-free production of integral membrane proteins in a soluble and folded state: Comparison with detergent micelles, bicelles and liposomes

Production of integral membrane proteins (IMPs) in a folded state is a key prerequisite for their functional and structural studies. In cell-free (CF) expression systems membrane mimicking components could be added to the reaction mixture that promotes IMP production in a soluble form. Here lipid-protein nanodiscs (LPNs) of different lipid compositions (DMPC, DMPG, POPC, POPC/DOPG) have been compared with classical membrane mimicking media such as detergent micelles, lipid/detergent bicelles and liposomes by their ability to support CF synthesis of IMPs in a folded and soluble state. Three model membrane proteins of different topology were used: homodimeric transmembrane (TM) domain of human receptor tyrosine kinase ErbB3 (TM-ErbB3, 1TM); voltage-sensing domain of K(+) channel KvAP (VSD, 4TM); and bacteriorhodopsin from Exiguobacterium sibiricum (ESR, 7TM). Structural and/or functional properties of the synthesized proteins were analyzed. LPNs significantly enhanced synthesis of the IMPs in a soluble form regardless of the lipid composition. A partial disintegration of LPNs composed of unsaturated lipids was observed upon co-translational IMP incorporation. Contrary to detergents the nanodiscs resulted in the synthesis of ~80% active ESR and promoted correct folding of the TM-ErbB3. None of the tested membrane mimetics supported CF synthesis of correctly folded VSD, and the protocol of the domain refolding was developed. The use of LPNs appears to be the most promising approach to CF production of IMPs in a folded state. NMR analysis of (15)N-Ile-TM-ErbB3 co-translationally incorporated into LPNs shows the great prospects of this membrane mimetics for structural studies of IMPs produced by CF systems.

Role of the nucleus in apoptosis: signaling and execution

Since their establishment in the early 1970s, the nuclear changes upon apoptosis induction, such as the condensation of chromatin, disassembly of nuclear scaffold proteins and degradation of DNA, were, and still are, considered as the essential steps and hallmarks of apoptosis. These are the characteristics of the execution phase of apoptotic cell death. In addition, accumulating data clearly show that some nuclear events can lead to the induction of apoptosis. In particular, if DNA lesions resulting from deregulation during the cell cycle or DNA damage induced by chemotherapeutic drugs or viral infection cannot be efficiently eliminated, apoptotic mechanisms, which enable cellular transformation to be avoided, are activated in the nucleus. The functional heterogeneity of the nuclear organization allows the tight regulation of these signaling events that involve the movement of various nuclear proteins to other intracellular compartments (and vice versa) to initiate and govern apoptosis. Here, we discuss how these events are coordinated to execute apoptotic cell death.

Post-translational Modification of Caspases: The Other Side of Apoptosis Regulation

Apoptosis is a crucial program of cell death that controls development and homeostasis of multicellular organisms. The main initiators and executors of this process are the Cysteine-dependent ASPartate proteASES - caspases. A number of regulatory circuits tightly control caspase processing and activity. One of the most important, yet, at the same time still poorly understood control mechanisms of activation of caspases involves their post-translational modifications. The addition and/or removal of chemical groups drastically alters the catalytic activity of caspases or stimulates their nonapoptotic functions. In this review, we will describe and discuss the roles of key caspase modifications such as phosphorylation, ubiquitination, nitrosylation, glutathionylation, SUMOylation, and acetylation in the regulation of apoptotic cell death and cell survival.

Cell death controlling complexes and their potential therapeutic role

Programmed cell death plays a central role in the regulation of homeostasis and development of multicellular organisms. Deregulation of programmed cell death is connected to a number of disorders, including cancer and autoimmune diseases. Initiation of cell death occurs in the multiprotein complexes or high molecular weight platforms. Composition, structure, and molecular interactions within these platforms influence the cellular decision toward life or death and, therefore, define the induction of a particular cell death program. Here, we discuss in detail the key cell-death complexes—including DISC, complex II, and TNFRI complex I/II, and the necrosome, RIPoptosome, apoptosome, and PIDDosome—that control apoptosis or necroptosis pathways as well as their regulation. The possibility of their pharmacological targeting leading to the development of new strategies of interference with cell death programs via control of the high molecular weight platforms will be discussed.

Caloric restriction - A promising anti-cancer approach: From molecular mechanisms to clinical trials

Cancer is the second leading cause of death worldwide and the morbidity is growing in developed countries. According to WHO, >14 million people per year are diagnosed with cancer and about 8 million die. Anti-cancer strategy includes chemo-, immune- and radiotherapy or their combination. Unfortunately, these widely used strategies often have insufficient efficacy and significant toxic effects on healthy cells. Consequently, the improvement of treatment approaches is an important goal. One of promising schemes to enhance the effect of therapy is the restriction of calorie intake or some nutrients. The combination of caloric restriction or its chemical mimetics along with anti-cancer drugs may suppress growth of tumor cells and enhance death of cancer cells. That will allow the dose of therapeutic drugs to be decreased and their toxic effects to be reduced. Here the possibility of using this combinatory therapy as well as the molecular mechanisms underlying this approach will be discussed.

Development and optimization of a coupled cell-free system for the synthesis of the transmembrane domain of the receptor tyrosine kinase ErbB3

A coupled cell-free expression system (CECF) for the production of the transmembrane domain of the human receptor tyrosine kinase ErbB3 (residues from 632 to 675) has been developed based on the Escherichia coli S30 extract. The synthesis of the domain in the soluble form in the presence of various detergents and in the form of an insoluble precipitate of the reaction mixture has been examined. The conditions for the purification of the recombinant domain obtained using the two approaches have been determined. The final yield of the target protein under optimal conditions was 1.8–2.0 mg per 1 ml of the reaction mixture.

Identification of new complex for caspase-2 activation after DNA damage

Caspase-2 is reported to play an initiator role in apoptotic cell death in response to DNA damage. In this study, the mechanism of caspase-2 activation after DNA damage was investigated in human ovarian cancer cells Caov-4 treated with the chemotherapeutic agent cisplatin. To isolate the protein complex that might be involved in caspase-2 activation, a combination of gel filtration and immunoprecipitation was used. In the first step the high molecular weight complexes were separated from caspase-2 monomers by means of gel-filtration and in the second step immunoprecipitation from the high molecular weight gel-filtration fractions allowed us to isolate the complex that contains caspase-2. Interestingly, this complex did not contain the protein RAIDD that is a core component of the PIDDosome platform; the latter was shown to play an essential role in DNA damage-induced caspase-2 activation. Finally, catalytically active caspase-2 was detected in this complex, which indicates the possibility of formation of an alternative platform for caspase2 activation in DNA damage-induced apoptosis.

Nutrient restriction in combinatory therapy of tumors

The main objective of anticancer treatment is the elimination of degenerated cells by the induction of programmed cell death. Various chemotherapy drugs and radiation are able to activate cell death mechanisms in tumors. However, unfortunately, monotherapy will always be insufficiently effective because of the variety and virulence of tumors, as well as their ability to develop resistance to drugs. Moreover, monotherapy might constrain many negative side effects. Therefore, the combination of different approaches and/or drugs will increase the efficiency of treatment. One such promising approach is the combination of nutrient restriction (NR) and various chemotherapeutic drugs. This approach may not only affect the autophagy but also influence apoptotic cell death. This review is focused on the potential of NR use in anticancer therapy, as well as the molecular mechanisms underlying this approach.

Mechanism of caspase-2 activation upon DNA damage.

The mechanism of caspase-2 activation in response to DNA damage was studied using human ovarian cancer cells Caov-4 treated with chemotherapeutic agent cisplatin. It was shown that mutations of the three cleavage sites of caspase-2 do not affect the assembly of the macromolecular complex of caspase-2 and its activation, but, conversely, stabilize this complex, most likely, via the inhibition of the dissociation of the active caspase-2.

Alterations in the nucleocytoplasmic transport in apoptosis: Caspases lead the way

Apoptosis is a mode of regulated cell death that is indispensable for the morphogenesis, development and homeostasis of multicellular organisms. Caspases are cysteine‐dependent aspartate‐specific proteases, which function as initiators and executors of apoptosis. Caspases are cytosolic proteins that can cleave substrates located in different intracellular compartments during apoptosis. Many years ago, the involvement of caspases in the regulation of nuclear changes, a hallmark of apoptosis, was documented. Accumulated data suggest that apoptosis‐associated alterations in nucleocytoplasmic transport are also linked to caspase activity. Here, we aim to discuss the current state of knowledge regarding this process. Particular attention will be focused on caspase nuclear entry and their functions in the demolition of the nucleus upon apoptotic stimuli.