In sarcomeres, α-actinin crosslinks actin filaments and anchors them to the Z-disk.FATZ proteins interact with α-actinin and five other core Z-disk proteins, contributing to myofibril assembly and maintenance as a protein interaction hub.Here we report the first structure and its cellular validation of α-actinin-2 in complex with a Z-disk partner, FATZ-1, which is best described as a conformational ensemble.We show that FATZ-1 forms a tight fuzzy complex with α-actinin-2 and propose a molecular interaction mechanism via main molecular recognition elements and secondary binding sites.The obtained integrative model reveals a polar architecture of the complex which, in combination with FATZ-1 multivalent scaffold function, might organise interaction partners and stabilise α-actinin-2 preferential orientation in the Z-disk.Finally, we uncover FATZ-1 ability to phase-separate and form biomolecular condensates with α-actinin-2, raising the intriguing question whether FATZ proteins can create an interaction hub for Z-disk proteins through membrane-less compartmentalization during myofibrillogenesis.

In sarcomeres, α-actinin cross-links actin filaments and anchors them to the Z-disk. FATZ (filamin-, α-actinin-, and telethonin-binding protein of the Z-disk) proteins interact with α-actinin and other core Z-disk proteins, contributing to myofibril assembly and maintenance. Here, we report the first structure and its cellular validation of α-actinin-2 in complex with a Z-disk partner, FATZ-1, which is best described as a conformational ensemble. We show that FATZ-1 forms a tight fuzzy complex with α-actinin-2 and propose an interaction mechanism via main molecular recognition elements and secondary binding sites. The obtained integrative model reveals a polar architecture of the complex which, in combination with FATZ-1 multivalent scaffold function, might organize interaction partners and stabilize α-actinin-2 preferential orientation in Z-disk. Last, we uncover FATZ-1 ability to phase-separate and form biomolecular condensates with α-actinin-2, raising the question whether FATZ proteins can create an interaction hub for Z-disk proteins through membraneless compartmentalization during myofibrillogenesis.

Actinin-2 plays a pivotal role in Z-disk assembly and stability as it crosslinks actin filaments from adjacent sarcomeres and acts as a binding platform for a number of Z-disk proteins.Among them is FATZ-1, a 30-kDa protein that appears when Z-bodies, the precursors of Z-disks, are formed.Accordingly, FATZ-1 is believed to have a central role at initial stages of myofibrillogenesis by serving as focal point for interactions with Z-disk proteins, but its structure and function remain unknown.Here, we used bioinformatics, CD and SAXS to show that FATZ-1 is intrinsically disordered after generating a soluble construct (D91 FATZ-1).We next studied the affinity and binding stoichiometry of a-actinin-2/FATZ-1 complex by ITC and SEC-MALS, and mapped down a shortest construct (mini FATZ-1) using XL/MS, NMR and LP/MS, as the disordered nature of this protein was a challenge for our crystallographic studies.We then managed to solve the structure of -actinin-2 rod/mini FATZ-1 complex at 2.7Å and -actinin-2 half dimer/D91 FATZ-1 complex at 3.2Å using MR and Se-Met variants of FATZ-1.In addition, we modelled the flexible regions of D91 FATZ-1 in complex with -actinin-2 by SAXS to have a complete molecular model of the complex.Finally, we studied FATZ-1 capacity to phase separate and form liquid droplets in the presence of -actinin-2.Together, our results provide a complete and detailed structural model of the -actinin-2/ FATZ-1 fuzzy complex as well as a plausible model on how FATZ might attract Z-disk proteins and form membrane-less compartmentalization at initial stages of myofibrillogenesis.

The degradation of misfolded, ubiquitinated proteins is essential for cellular homeostasis. These proteins are primarily degraded by the ubiquitin-proteasome system (UPS) and macroautophagy/autophagy serves as a backup mechanism when the UPS is overloaded. How autophagy and the UPS are coordinated is not fully understood. During the autophagy of misfolded, ubiquitinated proteins, referred to as aggrephagy, substrate proteins are clustered into larger structures in a SQSTM1/p62-dependent manner before they are sequestered by phagophores, the precursors to autophagosomes. We have recently shown that SQSTM1/p62 and ubiquitinated proteins spontaneously phase separate into micrometer-sized clusters in vitro. This enabled us to characterize the properties of the ubiquitin-positive substrates that are necessary for the SQSTM1/p62-mediated cluster formation. Our results suggest that aggrephagy is triggered by the accumulation of substrates with multiple ubiquitin chains and that the process can be inhibited by active proteasomes.

The removal of misfolded, ubiquitinated proteins is an essential part of the protein quality control. The ubiquitin-proteasome system (UPS) and autophagy are two interconnected pathways that mediate the degradation of such proteins. During autophagy, ubiquitinated proteins are clustered in a p62-dependent manner and are subsequently engulfed by autophagosomes. However, the nature of the protein substrates targeted for autophagy is unclear. Here, we developed a reconstituted system using purified components and show that p62 and ubiquitinated proteins spontaneously coalesce into larger clusters. Efficient cluster formation requires substrates modified with at least two ubiquitin chains longer than three moieties and is based on p62 filaments cross-linked by the substrates. The reaction is inhibited by free ubiquitin, K48-, and K63-linked ubiquitin chains, as well as by the autophagosomal marker LC3B, suggesting a tight cross talk with general proteostasis and autophagosome formation. Our study provides mechanistic insights on how substrates are channeled into autophagy.

Poly(ADP-ribose) glycohydrolase (PARG) regulates cellular poly(ADP-ribose) (PAR) levels by rapidly cleaving glycosidic bonds between ADP-ribose units. PARG interacts with proliferating cell nuclear antigen (PCNA) and is strongly recruited to DNA damage sites in a PAR- and PCNA-dependent fashion. Here we identified PARG acetylation site K409 that is essential for its interaction with PCNA, its localization within replication foci and its recruitment to DNA damage sites. We found K409 to be part of a non-canonical PIP-box within the PARG disordered regulatory region. The previously identified putative N-terminal PIP-box does not bind PCNA directly but contributes to PARG localization within replication foci. X-ray structure and MD simulations reveal that the PARG non-canonical PIP-box binds PCNA in a manner similar to other canonical PIP-boxes and may represent a new type of PIP-box. While the binding of previously described PIP-boxes is based on hydrophobic interactions, PARG PIP-box binds PCNA via both stabilizing hydrophobic and fine-tuning electrostatic interactions. Our data explain the mechanism of PARG-PCNA interaction through a new PARG PIP-box that exhibits non-canonical sequence properties but a canonical mode of PCNA binding.

1 Austrianova Biotechnology GmbH, Veterinärplatz 1, A-1210 Vienna, Austria 2 Children’s Cancer Research Institute, Kinderspitalgasse 6, A-1090 Vienna, Austria 3 Research Institute of Virology and Biomedicine, University of Veterinary Medicine, Veterinärplatz 1, A-1210 Vienna, Austria 4 Department of Pathobiology, University of Veterinary Medicine, Veterinärplatz 1, A-1210 Vienna, Austria 5 Department of Pharmacology and Toxicology, University of Vienna, Althanstraße 14, A-1090 Vienna, Austria

In vitro cultured mammalian cells are able to sense micro- and nanotopographical features of their substratum, resulting in contact guidance of the cells along topographical features, altered motility, proliferation and differentiation. We here used embossed polyester films with nanogrooves to study the dynamics of cell spreading and alignment of chinese hamster ovary (CHO) cells and cells of a rat myogenic cell line. Our results suggest that the response of the cells studied is driven by initial cytoplasmic protrusions, which emerge along the nanogrooves and pull the cells along the direction of the nanogrooves.

We used mechanically embossed polyester films to analyze the dynamics of cell alignment and cell-specific factors modulating the response of Chinese hamster ovary (CHO) cells and of a rat myogenic cell line to the surface topography. The films used had grooves with a periodicity of approximately 750 nm and a depth of 150 nm. Both cell lines responded to the topographical feature. On unpatterned control areas, cells of both lines showed a random distribution with orientation angles close to 45∘. Both cell types exhibited an elongated morphology on the patterned surface. CHO cells typically showed bipolar spreading. Their contact area increased almost exclusively along the groove direction. Likewise, freshly seeded rat myoblasts displayed protrusions emerging in parallel with the grooves. However, myoblasts frequently had more than two sites with plasma protrusions pulling the cells along different grooves. They could also develop lamellipodia expanding without a preferred direction and long filopodia.

We expressed the stachyose synthase from adzuki bean (Vigna angularis) in Arabidopsis thaliana, under the control of the constitutive CaMV 35S promoter. Transgenic lines had only trace amounts of stachyose under normal growth conditions but accumulated stachyose to similar levels as raffinose upon cold acclimation. Stachyose production did not alter the freezing tolerance of cold acclimated rosette leaves. Non-aqueous fractionation of sub-cellular compartments revealed that in cold acclimated plants, raffinose but not stachyose accumulated to a proportion higher than the compartment size fraction in the plastids. Since both oligosaccharides are synthesized in the cytosol, this provides evidence that the so far unknown raffinose transporter of the Arabidopsis chloroplast envelope does not efficiently transport stachyose. The failure of stachyose to influence freezing tolerance in Arabidopsis supports the hypothesis that raffinose family oligosaccharides might function in protecting the thylakoid but not the plasma membrane during freezing.

Voltage dependence and kinetics of activation of CaV1.2 channels are affected by structural changes in pore lining S6 segments of the α1-subunit. Significant effects on channel activation are induced by either proline or threonine substitutions in the lower third of segment IIS6 ('bundle crossing region'). Here we report that S435P in IS6 results in a large shift of the activation (−26mV) curve and slowed current kinetics. Threonine substitutions in positions Leu429 and Leu434 induced a similar kinetic phenotype with shifted activation curves. Double mutations in segments IS6 and IIS6 induced additive shifts of the activation curves, e.g.: L429T/I781T (-44.0±1.0), L434T/I781T (-50.3±0.8), L429T/L779T (-22.5±0.8) and L434T/L779T (-32.3±0.8). If the gating sensitive residues in the two neighboring segments IS6 and IIS6 do not interact then the change in free energy (ΔGdouble) of the double mutant is equal to the sum of the changes in free energy of the two single mutations (ΔGmut IS6 and ΔGmut IIS6 see scheme, see also Horovitz 1996). Double mutant cycle analysis revealed that the studied IS6 and IIS6 mutations are energetically independent and thus have independent impacts on activation gating. Supported by FWF-Project P19614-B11.

Voltage dependence and kinetics of CaV1.2 activation are affected by structural changes in pore-lining S6 segments of the α1-subunit. Significant effects are induced by either proline or threonine substitutions in the lower third of segment IIS6 (“bundle crossing region”), where S6 segments are likely to seal the channel in the closed conformation (Hohaus, A., Beyl, S., Kudrnac, M., Berjukow, S., Timin, E. N., Marksteiner, R., Maw, M. A., and Hering, S. (2005) J. Biol. Chem. 280, 38471–38477). Here we report that S435P in IS6 results in a large shift of the activation curve (-25.9 ± 1.2 mV) and slower current kinetics. Threonine substitutions at positions Leu-429 and Leu-434 induced a similar kinetic phenotype with shifted activation curves (L429T by -6.6 ± 1.2 and L434T by -12.1 ± 1.7 mV). Inactivation curves of all mutants were shifted to comparable extents as the activation curves. Interdependence of IS6 and IIS6 mutations was analyzed by means of mutant cycle analysis. Double mutations in segments IS6 and IIS6 induce either additive (L429T/I781T, -34.1 ± 1.4 mV; L434T/I781T, -40.4 ± 1.3 mV; L429T/L779T, -12.6 ± 1.3 mV; and L434T/L779T, -22.4 ± 1.3 mV) or nonadditive shifts of the activation curves along the voltage axis (S435P/I781T, -33.8 ± 1.4 mV). Mutant cycle analysis revealed energetic coupling between residues Ser-435 and Ile-781, whereas other paired mutations in segments IS6 and IIS6 had independent effects on activation gating.

Gene directed-enzyme prodrug therapy (GDEPT) is an approach for sensitization of tumor cells to an enzymatically activated, otherwise nontoxic, prodrug. Cytochrome P450 2B1 (CYP2B1) metabolizes the prodrugs cyclophosphamide (CPA) and ifosfamide (IFA) to produce the cytotoxic substances phosphoramide mustard and isophosphoramide mustard as well as the byproduct acrolein. We have constructed a retroviral promoter conversion (ProCon) vector for breast cancer GDEPT. The vector allows expression of CYP2B1 from the mouse mammary tumor virus (MMTV) promoter known to be active in the mammary glands of transgenic animals. It is anticipated to be used for the generation of encapsulated viral vector producing cells which, when placed inside or close to a tumor, will act as suppliers of the therapeutic CYP2B1 protein as well as of the therapeutic vector itself. The generated vector was effectively packaged by virus producing cells and allowed the production of high levels of enzymatically active CYP2B1 in infected cells which sensitized them to killing upon treatment with both IFA and CPA. Determination of the respective IC(50) values demonstrated that the effective IFA dose was reduced by sixteen folds. Infection efficiencies in vivo were determined using a reporter gene-bearing vector in a mammary cancer cell-derived xenograft tumor mouse model.

Biomaterial surface chemistry and nanoscale topography are important for many potential applications in medicine and biotechnology as they strongly influence cell function, adhesion and proliferation. In this work, we present periodic surface structures generated by linearly polarized KrF laser light (248 nm) on polystyrene (PS) foils. These structures have a periodicity of 200–430 nm and a depth of 30–100 nm, depending on the angle of incidence of the laser beam. The changes in surface topography and chemistry were analysed by atomic force microscopy (AFM), advancing water contact-angle measurements, Fourier-transform infrared spectroscopy using an attenuated total reflection device (ATR-FTIR) and X-ray photoelectron spectroscopy (XPS). We show that the surface laser modification results in a significantly enhanced adhesion and proliferation of human embryonic kidney cells (HEK-293) compared to the unmodified polymer foil. Furthermore, we report on the alignment of HEK-293 cells, Chinese hamster ovary (CHO-K1) cells and skeletal myoblasts along the direction of the structures. The results indicate that the presence of nanostructures on the substrates can guide cell alignment along definite directions, and more importantly, in our opinion, that this alignment is only observed when the periodicity is above a critical periodicity value that is cell-type specific.

Both alkaline and acidic α-galactosidases (α-d-galactoside galactohydrolases, E.C.3.2.1.22) isolated from various plant species have been described, although little is known about their co-occurrence and functions in germinating seeds. Here, we report on the isolation of two cDNAs, encoding for α-galactosidases from maturing and germinating seeds of Pisum sativum. One was identified as a member of the acidic α-galactosidase of the family 27 glycosyl hydrolase cluster and the other as a member of the family of alkaline α-galactosidases, which are highly homologous to seed imbibition proteins (SIPs). PsGAL1 transcripts, encoding for the ACIDIC α-GALACTOSIDASE, were predominately expressed during seed maturation and acidic enzyme activities were already present in dry seeds, showing little changes during seed germination. Compartmentation studies revealed that acidic α-galactosidases were located in protein storage vacuoles (PSVs). PsAGA1, encoding for the ALKALINE α-GALACTOSIDASE, was only expressed after radicle protrusion, when about 50% of RFOs have already been broken down. RFO breakdown was markedly decreased when the translation of the alkaline enzyme was inhibited, providing evidence that PsAGA1 indeed functioned in RFO degradation. Based on these data, we present an integrated model of RFO breakdown by two sequentially active α-galactosidases in pea seeds.

The bacteriophage-encoded holin proteins are known to promote bacterial cell lysis by forming lesions within the cytoplasmic membrane. Recently, we have shown that the bacteriophage lambda-holin protein exerts cytotoxic activity also in eukaryotic cells accounting for a reduced tumour growth in vivo. In order to elucidate the mechanisms of lambda-holin-induced mammalian cell death, detailed biochemical and morphological analyses were performed. Colocalization analyses by subcellular fractionation and organelle-specific fluorescence immunocytochemistry indicated the presence of the lambda-holin protein in the endoplasmic reticulum and in mitochondria. Functional studies using the mitochondria-specific fluorochrome JC-1 demonstrated a loss of mitochondrial transmembrane potential in response to lambda-holin expression. Morphologically, these cells exhibited unfragmented nuclei but severe cytoplasmic vacuolization representing signs of oncosis/necrosis rather than apoptosis. Consistently, Western blot analyses indicated neither an activation of effector caspases 3 and 7 nor cleavage of the respective substrate poly(ADP-ribose) polymerase (PARP) in an apoptosis-specific manner. These findings suggest that the lambda-holin protein mediates a caspase-independent non-apoptotic mode of cell death.

We describe here the modification of various polymers (polytetrafluoroethylene, polyethyleneterephthalate, and polyvinyl alcohol) by UV-irradiation with wavelengths below 200 nm in an inert or reactive atmosphere. The light sources employed are F2- or excimer lasers and excimer lamps. The reactive gases include ammonia (NH3), acetylene (C2H2), and oxygen (O2). Photo-dissociated fragments of these gases can react with the polymers or be deposited thereon, resulting in new chemical groups at the surface. Special emphasis is put to improved adhesion of biological cells at these modified surfaces. Potentials applications include cell coated medical implants and prostheses as well as cell micro-arrays for high throughput screening.

Single-step methods for the generation of patterned surfaces on hydrogels are presented. Poly(vinyl alcohol) films covalently bonded on glass cover slips and commercially available hydrogel-coated polystyrene plates were used as cell-repellent surfaces. Cell-adhesive domains were created by spotting dilute solutions of sodium hypochlorite onto the surfaces. Alternatively, domains supporting cell attachment were created by exposure to UV light from a xenon excimer lamp, employing a contact mask. Rat skeletal myoblast cells, HEK 293 human embryonic kidney cells and Caco-2 colon carcinoma cells adhered and spread exclusively on modified areas. The surfaces are durable for weeks under cell culture conditions and re-usable after removal of the cells by trypsin treatment. Arrays of adhesive spots seeded with cells at a low density permitted dynamic monitoring of cell proliferation. Selected colonies can be harvested from the surfaces by means of local trypsination. Thus, these techniques may provide useful tools for the isolation of clonal cell populations. Additionally, we demonstrate the possibility of surface-mediated gene delivery from the micro patterns. We show that DNA, complexed with a lipid reagent, can be adsorbed on modified poly(vinyl alcohol) coatings, resulting in spatially controlled adhesion and reverse transfection of HEK 293 cells.

The hexasaccharide ajugose, alpha-D-galactopyranosyl-(1-->6)-alpha-D-galactopyranosyl-(1-->6)-O-alpha-D-galactopyranosyl-(1-->6)-alpha-D-galactopyranosyl-(1-->6)-alpha-D-glucopyranosyl-(1<-->2)-beta-D-fructofuranoside, generally uncommon in legumes, was detected in the seeds of Vigna mungo L. by TLC and paper chromatography. Ajugose was then isolated by silica gel chromatography and its structure was established by acid and enzymatic hydrolysis, fast atom bombardment mass spectrometry and both one- and two-dimensional 1H and 13C NMR techniques.

L-Arabinitol 4-dehydrogenase (Lad1) of the cellulolytic and hemicellulolytic fungus Hypocrea jecorina (anamorph: Trichoderma reesei) has been implicated in the catabolism of L-arabinose, and genetic evidence also shows that it is involved in the catabolism of D-xylose in xylitol dehydrogenase (xdh1) mutants and of D-galactose in galactokinase (gal1) mutants of H. jecorina. In order to identify the substrate specificity of Lad1, we have recombinantly produced the enzyme in Escherichia coli and purified it to physical homogeneity. The resulting enzyme preparation catalyzed the oxidation of pentitols (L-arabinitol) and hexitols (D-allitol, D-sorbitol, L-iditol, L-mannitol) to the same corresponding ketoses as mammalian sorbitol dehydrogenase (SDH), albeit with different catalytic efficacies, showing highest k(cat)/K(m) for L-arabinitol. However, it oxidized galactitol and D-talitol at C4 exclusively, yielding L-xylo-3-hexulose and D-arabino-3-hexulose, respectively. Phylogenetic analysis of Lad1 showed that it is a member of a terminal clade of putative fungal arabinitol dehydrogenase orthologues which separated during evolution of SDHs. Juxtapositioning of the Lad1 3D structure over that of SDH revealed major amino acid exchanges at topologies flanking the binding pocket for d-sorbitol. A lad1 gene disruptant was almost unable to grow on L-arabinose, grew extremely weakly on L-arabinitol, D-talitol and galactitol, showed reduced growth on D-sorbitol and D-galactose and a slightly reduced growth on D-glucose. The weak growth on L-arabinitol was completely eliminated in a mutant in which the xdh1 gene had also been disrupted. These data show not only that Lad1 is indeed essential for the catabolism of L-arabinose, but also that it constitutes an essential step in the catabolism of several hexoses; this emphasizes the importance of such reductive pathways of catabolism in fungi.