Proc Natl Acad Sci U S A. 2009 Mar 24;106(12):4805-9.

Sessile hemocytes as a hematopoietic compartment in Drosophila melanogaster.

Márkus R, Laurinyecz B, Kurucz E, Honti V, Bajusz I, Sipos B, Somogyi K, Kronhamn J, Hultmark D, Andó I Institute of Genetics, Biological Research Center of the Hungarian Academy of Sciences, PO Box 521, H-6701, Szeged, Hungary

The blood cells, or hemocytes, in Drosophila participate in the immune response through the production of antimicrobial peptides, the phagocytosis of bacteria, and the encapsulation of larger foreign particles such as parasitic eggs; these immune reactions are mediated by phylogenetically conserved mechanisms. The encapsulation reaction is analogous to the formation of granuloma in vertebrates, and is mediated by large specialized cells, the lamellocytes. The origin of the lamellocytes has not been formally established, although it has been suggested that they are derived from the lymph gland, which is generally considered to be the main hematopoietic organ in the Drosophila larva. However, it was recently observed that a subepidermal population of sessile blood cells is released into the circulation in response to a parasitoid wasp infection. We set out to analyze this phenomenon systematically. As a result, we define the sessile hemocytes as a novel hematopoietic compartment, and the main source of lamellocytes.

The Plant Cell, 2008 Oct. Vol. 20: 2552-2557

The three-dimensional network of the thylakoid membranes in plants: Quasi helical model of the granum-stroma assembly

L. Mustárdy¹, K. Buttle², G. Steinbach1, C. Mannella² and G. Garab^1
1Institute of Plant Biology, Biological Research Center, Hungarian Academy of Sciences, H-6701 Szeged, Box 521, Hungary; ² Resource for Visualization of Biological Complexity, Wadsworth Center, Empire State Plaza, Box 509, Albany, NY 12201-0509, USA

We investigated the three-dimensional architecture of isolated granal thylakoid membranes by using high voltage electron tomography on 250 nm thick sections of isolated intact thylakoid preparations fixed and stained with conventional electron microscopy techniques. High resolution reconstructed tomographic images were obtained, which clearly resolved the membranes and the lumenal spaces, and thus also the connections between the stacked and non-stacked membranes. Based on the series of tomographic sections and the computer assisted 3D models, and also the using the electron tomography data of chloroplasts within cryo-immobilized, freeze substituted lettuce leaves (Shimoni et al. 2005, Plant Cell 17: 2580), we propose a refined model of the granum stroma assembly, in particular on the nature and origin of the granum-stroma junctions and the assembly of the granal thylakoid membrane system. The model takes into account the following structural factors and membrane properties: (i) the size differences between the stroma exposed sides of the two photosystems, i.e., the fact that the stromal sides of photosystem II (PSII) and its main light harvesting complexes (LHCII) are flat, while PSI and the ATP synthase protrude significantly into the stroma liquid; (ii) the ability of LHCII and PSII to self-assemble into large domains, i.e., the ability of LHCII and PSII, and PSI and the ATP synthase for lateral segregation; (iii) the stacking of membranes possessing flat membrane surfaces, which stabilizes the lateral segregation of the (super)complexes (iv) the fusion and overlap of membranes during the growth of membranes. These factors might bring about the self-assembly of a quasi-helical organization of the granum-stroma contiguous thylakoid membrane system that encloses a single interior aqueous phase.

Proc Natl Acad Sci U S A. 2008 Mar 11;105(10):3768-73.

Human HLTF functions as a ubiquitin ligase for proliferating cell nuclear antigen polyubiquitination.

Unk I*, Hajdú I*, Fátyol K, Hurwitz J, Yoon JH, Prakash L, Prakash S, Haracska L*
*Institute of Genetics, Biological Research Center, Hungarian Academy of Sciences, Szeged, Hungary.

Human helicase-like transcription factor (HLTF) is frequently inactivated in colorectal and gastric cancers. Here, we show that HLTF is a functional homologue of yeast Rad5 that promotes error-free replication through DNA lesions. HLTF and Rad5 share the same unique structural features, including a RING domain embedded within a SWI/SNF helicase domain and an HIRAN domain. We find that inactivation of HLTF renders human cells sensitive to UV and other DNA-damaging agents and that HLTF complements the UV sensitivity of a rad5Delta yeast strain. Also, similar to Rad5, HLTF physically interacts with the Rad6-Rad18 and Mms2-Ubc13 ubiquitin-conjugating enzyme complexes and promotes the Lys-63-linked polyubiquitination of proliferating cell nuclear antigen at its Lys-164 residue. A requirement of HLTF for error-free postreplication repair of damaged DNA is in keeping with its cancer-suppression role.

Proc Natl Acad Sci U S A. 2008 May 13;105(19):6888-93.

Terahertz radiation from bacteriorhodopsin reveals correlated primary electron and proton transfer processes

Groma GI*, Hebling J, Kozma IZ, Váró G*, Hauer J, Kuhl J, Riedle E.
*Institute of Biophysics, Biological Research Center of the Hungarian Academy of Sciences, H-6726, Szeged, Hungary

The kinetics of electrogenic events associated with the different steps of the light-induced protonpumpof bacteriorhodopsin is well studied in a wide range of time scales by direct electric methods. However, the investigation of the fundamental primary charge translocation phenomena taking place in the functional energy conversion process of this protein, and in other biomolecular assemblies using light energy, has remained experimentally unfeasible because of the lack of proper detection technique operating in the 0.1- to 20-THz region. Here, we show that extending the concept of the familiar Hertzian dipole emission into the extreme spatial and temporal range of intramolecular polarization processes provides an alternative way to study ultrafast electrogenic events on naturally ordered biological systems. Applying a relatively simple experimental arrangement based on this idea, we were able to observe light-induced coherent terahertz radiation from bacteriorhodopsin with femtosecond time resolution. The detected terahertz signal was analyzed by numerical simulation in the framework of different models for the elementary polarization processes. It was found that the principal component of the terahertz emission can be well described by excited-state intramolecular electron transfer within the retinal chromophore. An additional slower process is attributed to the earliest phase of the proton pump, probably occurring by the redistribution of a H bond near the retinal. The correlated electron and proton translocation supports the concept, assigning a functional role to the light-induced sudden polarization in retinal proteins.

Mol Cell. 2007 Oct 12;28(1):167-75.

Yeast Rad5 protein required for postreplication repair has a DNA helicase activity specific for replication fork regression.

Blastyák A, Pintér L, Unk I, Prakash L, Prakash S, Haracska L.
Institute of Genetics, Biological Research Center, Hungarian Academy of Sciences, Szeged, Temesvari krt.62, H-6726, Hungary.

Lesions in the template DNA strand block the progression of the replication fork. In the yeast Saccharomyces cerevisiae, replication through DNA lesions is mediated by different Rad6-Rad18-dependent means, which include translesion synthesis and a Rad5-dependent postreplicational repair pathway that repairs the discontinuities that form in the DNA synthesized from damaged templates. Although translesion synthesis is well characterized, little is known about the mechanisms that modulate Rad5-dependent postreplicational repair. Here we show that yeast Rad5 has a DNA helicase activity that is specialized for replication fork regression. On model replication fork structures, Rad5 concertedly unwinds and anneals the nascent and the parental strands without exposing extended single-stranded regions. These observations provide insight into the mechanism of postreplicational repair in which Rad5 action promotes template switching for error-free damage bypass.

Curr Biol. 2007 Apr 3;17(7):649-54.

Nimrod, a putative phagocytosis receptor with EGF repeats in Drosophila plasmatocytes.

Kurucz E, Márkus R, Zsámboki J, Folkl-Medzihradszky K, Darula Z, Vilmos P, Udvardy A, Krausz I, Lukacsovich T, Gateff E, Zettervall CJ, Hultmark D, Andó I.
Institute of Genetics, Biological Research Center of the Hungarian Academy of Sciences, H-6701 Szeged, Hungary.

The hemocytes, the blood cells of Drosophila, participate in the humoral and cellular immune defense reactions against microbes and parasites [1-8]. The plasmatocytes, one class of hemocytes, are phagocytically active and play an important role in immunity and development by removing microorganisms as well as apoptotic cells. On the surface of circulating and sessile plasmatocytes, we have now identified a protein, Nimrod C1 (NimC1), which is involved in the phagocytosis of bacteria. Suppression of NimC1 expression in plasmatocytes inhibited the phagocytosis of Staphylococcus aureus. Conversely, overexpression of NimC1 in S2 cells stimulated the phagocytosis of both S. aureus and Escherichia coli. NimC1 is a 90-100 kDa single-pass transmembrane protein with ten characteristic EGF-like repeats (NIM repeats). The nimC1 gene is part of a cluster of ten related nimrod genes at 34E on chromosome 2, and similar clusters of nimrod-like genes are conserved in other insects such as Anopheles and Apis. The Nimrod proteins are related to other putative phagocytosis receptors such as Eater and Draper from D. melanogaster and CED-1 from C. elegans. Together, they form a superfamily that also includes proteins that are encoded in the human genome.

J Cell Sci. 2007 Sep 15;120(Pt 18):3238-48.

Structurally related TPR subunits contribute differently to the function of the anaphase-promoting complex in Drosophila melanogaster

Margit Pál, Olga Nagy, Dalma Ménesi, Andor Udvardy and Péter Deák
Institute of Biochemistry, Biological Research Center, H-6726 Szeged, Hungary

The anaphase-promoting complex/cyclosome or APC/C is a key regulator of chromosome segregation and mitotic exit in eukaryotes. It contains at least 11 subunits, most of which are evolutionarily conserved. The most abundant constituents of the vertebrate APC/C are the four structurally related tetratrico-peptide repeat (TPR) subunits, the functions of which are not yet precisely understood. Orthologues of three of the TPR subunits have been identified in Drosophila. We have shown previously that one of the TPR subunits of the Drosophila APC/C, Apc3 (also known as Cdc27 or Mákos), is essential for development, and perturbation of its function results in mitotic cyclin accumulation and metaphase-like arrest. In this study we demonstrate that the Drosophila APC/C associates with a new TPR protein, a genuine orthologue of the vertebrate Apc7 subunit that is not found in yeasts. In addition to this, transgenic flies knocked down for three of the TPR genes Apc6 (Cdc16), Apc7 and Apc8 (Cdc23), by RNA interference were established to investigate their function. Whole-body expression of subunit- specific dsRNA efficiently silences these genes resulting in only residual mRNA concentrations. Apc6/Cdc16 and Apc8/Cdc23 silencing induces developmental delay and causes different pupal lethality. Cytological examination showed that these animals had an elevated level of apoptosis, high mitotic index and delayed or blocked mitosis in a prometaphase- metaphase-like state with overcondensed chromosomes. The arrested neuroblasts contained elevated levels of Cyclin B but, surprisingly, Cyclin A appeared to be degraded normally. Contrary to the situation for the Apc6/Cdc16 and Apc8/Cdc23 genes, the apparent loss of Apc7 function does not lead to the above abnormalities. Instead, the Apc7 knocked down animals and null mutants are viable and fertile, although they display mild chromosome segregation defects and anaphase delay. Nevertheless, the Apc7 subunit shows synergistic genetic interaction with Apc8/Cdc23 that, together with the phenotypic data, assumes a limited functional role for Apc7. Taken together, these data suggest that the structurally related TPR subunits contribute differently to the function of the anaphase-promoting complex.

Proc Natl Acad Sci U S A. 2007 May 8;104(19):7945-50

Hyperfluidization-coupled membrane microdomain reorganization is linked to activation of the heat shock response in a murine melanoma cell line.

Nagy E, Balogi Z, Gombos I, Akerfelt M, Björkbom A, Balogh G, Török Z, Maslyanko A, Fiszer-Kierzkowska A, Lisowska K, Slotte PJ, Sistonen L, Horváth I, Vígh L.
Institute of Biochemistry, Biological Research Center, Hungarian Academy of Sciences, Temesvari krt. 62, Szeged, H-6701, Hungary.

Trends Biochem Sci. 2007 Aug;32(8):357-63

Can the stress protein response be controlled by 'membrane-lipid therapy'?

Vigh L, Horváth I, Maresca B, Harwood JL.
Institute of Biochemistry, Biological Research Center, Hungarian Academy of Sciences, Temesvari krt. 62, Szeged, H-6701, Hungary.

Due to their multiply and vital functions, stress protein molecular chaperones (heat shock proteins, Hsps) play a fundamental role in the pathology of several human diseaes. Aberrantly high level of certain Hsp classes is characteristic in cancer cells and the converse situation applies for type 2 diabetes or neurodegeneration. In accordance, understanding the mechanism whereby cells can elicit a stress protein response is of key importance. As a "central dogma" earlier it was suggested, that stress-induced protein denaturation serves as a primary stress-sensing machinery which triggers Hsp gene expression. From the past decade, a new but not exclusive cellular "thermosensor" model has evolved, which predicts the existence of membrane-associated stress sensing and signaling mechanisms. Changes in the physical state (fluidity) and/or composition of lipid molecular species with the concomitant destabilization/reorganization of membrane microdomains may serve as the "molecular switch" for the operation of these "cellular thermometers". Correcting the defects of membrane domains, engaged in the generation and transmission of stress signals may be of paramount importance for the design of new drugs with the ability to induce or attenuate the level of a particular class of heat shock proteins.

Chem Rev. 2007 Aug;107(8):3498-513

Systematic genome reductions: theoretical and experimental approaches

Fehér T, Papp B, Pál C, Pósfai G.
Institute of Biochemistry, Biological Research Center, Hungarian Academy of Sciences, Temesvari krt. 62, Szeged, H-6701, Hungary.

The living cell is the most complex structure, known to man, in the micrometer size range. On the road to understand its many and complex chemical reactions and exploit the enormous industrial potential it represents, a reductionist approach at the whole-cell level can clearly be beneficial. Even the simplest cell is made up of tens of millions of molecules of thousands of different kinds interacting in a complex cellular network. However, since the cell is a self-organizing entity with most of the cell's hereditary information needed for structure and function coded in its genes, cell simplification can be reduced to the task of engineering the genome. Genome reduction projects are motivated by both academic and industrial interests with the most fundamental questions of life at heart. How far can the simplification go? What is the minimal gene set needed for sustaining life in a defined environment? Can we reduce the genome to a point where all components and reactions can be fully catalogued and characterized? Such whole-scale reductions as well as bottom-up approaches of synthesizing entire genomes with the ultimate goal of building a living cell might be far off. However, on the practical side, improvement of certain cellular functions by streamlining genomes is existing reality.

Curr Biol. 2007 Sep 4;17(17):1456-64

Arabidopsis thaliana Circadian Clock Is Regulated by the Small GTPase LIP1.

Kevei E, Gyula P, Fehér B, Tóth R, Viczián A, Kircher S, Rea D, Dorjgotov D, Schäfer E, Millar AJ, Kozma-Bognár L, Nagy F.
Institute of Plant Biology, Biological Research Centre of the Hungarian Academy of Sciences, Temesvári krt. 62, Szeged H-6726, Hungary.

BACKGROUND: At the core of the eukaryotic circadian network, clock genes/proteins form multiple transcriptional/translational negative-feedback loops and generate a basic approximately 24 hr oscillation, which provides daily regulation for a wide range of processes. This temporal organization enhances the fitness of the organism only if it corresponds to the natural day/night cycles. Light is the most effective signal in synchronizing the oscillator to environmental cycles. RESULTS: The lip1-1 (light insensitive period 1) mutant isolated from the model plant Arabidopsis thaliana displays novel circadian phenotypes arising from specific defects in the light input pathway to the oscillator. In wild-type plants, period length shortens with increasing light fluence rates and the phase of rhythms can be shifted by light pulses administered to dark-adapted plants. In contrast, in lip1-1, period length is nearly insensitive to light intensity and significantly larger phase shifts (delays) can be induced during the subjective night. The mutant also displays elevated photomorphogenic responses to red and blue light, which cannot be explained by the circadian defect, suggesting distinct functions for LIP1 in the circadian light input and photomorphogenesis. The LIP1 gene encodes a functional, plant-specific atypical small GTPase, and therefore we postulate that it acts similarly to ZEITLUPE at postranscriptional level. CONCLUSIONS: LIP1 represents the first small GTPase implicated in the circadian system of plants. LIP1 plays a unique negative role in controlling circadian light input and is required for precise entrainment of the plant clock.

Nucleic Acids Res. 2006 May 10;34(9):2508-15.

Human Ape2 protein has a 3'-5' exonuclease activity that acts preferentially on mismatched base pairs.

Burkovics P , Szukacsov V , Unk I , Haracska L .
Institute of Genetics, Biological Research Center, Hungarian Academy of Sciences, Temesvari krt. 62, Szeged, H-6701, Hungary.

DNA damage is the reason for the formation of different types of cancers. Therefore it is essential to reduce the arising mutations in the genome, caused by the damaged DNA. All the participants of the repair processes can potentially be targets of new anticancer drugs.
Cellular DNA is subject to attack by a variety of agents of exogenous and endogenous origins. Oxidative attack on DNA by free radical species happens continuously during normal cellular metabolism, generating abasic sites (AP sites) and DNA strand breaks with modified 3’-termini, such as 3’-phosphate or 3’-phosphoglycolate (3’-PG). The non-coding AP-sites as well as modified 3’-DNA ends are inhibitory to synthesis by DNA polymerases, or if bypassed, are highly mutagenic; consequently, their repair is essential for retaining the stability of the genome. Class II AP endonucleases are able to function in the removal of this kind of DNA damage.
Ape2 is one of the human class II AP endonucleases, but the role and the biochemical properties of Ape2 have not been ascertained yet. In this study, we have clarified the enzymatic activities of human Ape2 and showed how it protects the integrity of the human genome.

Phys. Rev. Lett. 97, 058301 (2006)

Direct measurement of torque in the optical trap and its application to Double-Strand DNA

László Oroszi¹, Péter Galajda¹, Huba Kirei¹, Sándor Bottka², Pál Ormos¹

¹Institute of Biophysics, Biological Research Center, Hungarian Academy of Sciences, 6726 Szeged, Hungary
²Institute of Plant Biology, Biological Research Center, Hungarian Academy of Sciences, 6726 Szeged, Hungary

The researchers in the Institute of Biophysics developed a technique by which it is possible to directly measure torque exerted by single biopolymers such as dsDNA.

The technique is based on the rotational manipulation of flat microparticles in optical traps formed by linearly polarized light. The molecule under study is attached to the microparticle by one end and to a fixed surface (cover slide) by the other end. By rotating the polarization plane of the light the microparticle can be rotated and so the attached molecule can be twisted and torsionally stressed. The torque generated by the molecule is directly compared to the torque of the orienting light. The latter is determined from the rotational Brownian-fluctuations of the microparticle in the optical trap.

As a demonstration of the method the torsional stiffness (i.e. torsional modulus) of dsDNA was determined experimentally and compared to the results of polymer theories and other, more indirect experiments.