Order from disorder in the sarcomere: FATZ forms a fuzzy but tight complex and phase-separated condensates with α-actinin.

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.

Phasing out the bad-How SQSTM1/p62 sequesters ubiquitinated proteins for degradation by autophagy.

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.

p62 filaments capture and present ubiquitinated cargos for autophagy.

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.

A novel non-canonical PIP-box mediates PARG interaction with PCNA.

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.

Stachyose in the cytosol does not influence freezing tolerance of transgenic Arabidopsis expressing stachyose synthase from adzuki bean.

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.

Coupled and independent contributions of residues in IS6 and IIS6 to activation gating of CaV1.2.

Voltage dependence and kinetics of Ca(V)1.2 activation are affected by structural changes in pore-lining S6 segments of the alpha(1)-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.

Mouse mammary tumor virus promoter-containing retroviral promoter conversion vectors for gene-directed enzyme prodrug therapy are functional in vitro and in vivo.

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.

Enzymatic breakdown of raffinose oligosaccharides in pea seeds.

Both alkaline and acidic alpha-galactosidases (alpha-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 alpha-galactosidases from maturing and germinating seeds of Pisum sativum. One was identified as a member of the acidic alpha-galactosidase of the family 27 glycosyl hydrolase cluster and the other as a member of the family of alkaline alpha-galactosidases, which are highly homologous to seed imbibition proteins (SIPs). PsGAL1 transcripts, encoding for the ACIDIC alpha-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 alpha-galactosidases were located in protein storage vacuoles (PSVs). PsAGA1, encoding for the ALKALINE alpha-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 alpha-galactosidases in pea seeds.

Proliferation of aligned mammalian cells on laser-nanostructured polystyrene.

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.

Bacteriophage-encoded toxins: the lambda-holin protein causes caspase-independent non-apoptotic cell death of eukaryotic cells.

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.

Enniatin exerts p53-dependent cytostatic and p53-independent cytotoxic activities against human cancer cells.

Worldwide, multiple Fusarium mycotoxins occur as contaminants of cereals with important impacts on human and animal health. The aim of this study was to investigate the effects of the widespread Fusarium secondary metabolite enniatin (ENN), a cyclic hexadepsipeptide, on human cell growth and survival. While short-term exposure (up to 8 h) to ENN at nanomolar concentrations slightly but significantly stimulated cell proliferation, it showed profound apoptosis-inducing effects especially against various human cancer cell types at low micromolar concentrations (already after 24 h of treatment). Several cellular changes indicative for programmed cell death such as cell shrinkage, chromatin condensation, DNA fragmentation, and apoptotic body formation were observed. Correspondingly, the cleavage of poly(ADP-ribosyl)polymerase and the activation of multiple caspases accompanied a distinct loss of mitochondrial membrane potential. To investigate the impact of apoptosis- and cell cycle-regulating proteins on ENN activity, HCT116 cells with homozygously disrupted p53, p21, or bax genes were analyzed. In vitality assays, no significant influences of these proteins on the anticancer activity of ENN were detectable. In contrast, 3H-thymidine incorporation revealed a significantly more efficient block of DNA synthesis in p53 wild-type as compared to knock-out cells. Accordingly, fluorescence-activated cell sorting analysis demonstrated a stronger ENN-induced cell cycle arrest in the G0/G1 phase. Profound ENN-mediated induction of p53 and the p53-downstream cell cycle inhibitor p21 were detectable in p53 wild-type cells by Western blotting. P53-independent p21 induction was also detectable at higher ENN concentrations in p53 (-/-) cells. In contrast, bax activation by ENN was independent of the cellular p53 status. In summary, our results suggest that short-term exposure to very low ENN concentrations, for example, via food intake, might have tumor-promoting functions based on growth stimulation. In contrast, elevated ENN concentrations exert profound p53-dependent cytostatic and p53-independent cytotoxic activities especially against human cancer cells, suggesting a potential quality of ENN as an anticancer drug.

Inhibition of raffinose oligosaccharide breakdown delays germination of pea seeds.

Raffinose family oligosaccharides (RFOs) are almost ubiquitous in seeds and have been hypothesized to constitute an important energy source during germination. To test this hypothesis we applied a specific alpha-galactosidase inhibitor (1-deoxygalactonojirimycin, DGJ) to germinating pea seeds, resulting in a complete blocking of RFO breakdown. The germination rates of DGJ-treated seeds dropped drastically to about 25% of controls two days after imbibition. Similarly, the activities of the key enzymes in the galactose salvage pathway galactokinase, UDP-galactose pyrophosphorylase and UDP-galactose 4'-epimerase, were also significantly lower in seeds treated with the inhibitor. The inhibitory effect on germination could be relieved by galactose but only partially by sucrose, indicating that galactose, in addition to providing easily available energy for growth, may also be an important component of the sugar signaling pathway during germination. Taken together our study, for the first time, provides clear evidence that RFOs play an important role for early germination.

Simple and versatile methods for the fabrication of arrays of live mammalian cells.

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.

Isolation and structural analysis of ajugose from Vigna mungo L.

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.

myo-Inositol and sucrose concentrations affect the accumulation of raffinose family oligosaccharides in seeds.

Raffinose family oligosaccharides (RFOs) fulfil multiple functions in plants. In seeds, they possibly protect cellular structures during desiccation and constitute carbon reserves for early germination. Their biosynthesis proceeds by the transfer of galactose units from galactinol to sucrose. Galactinol synthase (GolS), which mediates the synthesis of galactinol from myo-inositol and UDP-galactose, has been proposed to be the key enzyme of the pathway. However, no significant relationship was detected between the extractable GolS activity and the amount of RFOs in seeds from seven pea (Pisum sativum L.) genotypes selected for high variation in RFO content. Instead, a highly significant correlation was found between the levels of myo-inositol and RFOs. Moderately strong relationships were also found between sucrose and RFO content as well as between myo-inositol and galactinol. Further evidence for a key role of myo-inositol for the synthesis of galactinol was obtained by feeding exogenous myo-inositol to intact pea seeds and by the analysis of four barley (Hordeum vulgare L.) low phytic acid mutants. In seeds of three of these mutants, the reduced demand for myo-inositol for the synthesis of phytic acid (myo-inositol 1,2,3,4,5,6-hexakisphosphate) was associated with an increased level in myo-inositol. The mutants seeds also contained more galactinol than wild-type seeds. The results suggest that the extent of RFO accumulation is controlled by the levels of the initial substrates, myo-inositol and sucrose, rather than by GolS activity alone.

The metabolic role and evolution of L-arabinitol 4-dehydrogenase of Hypocrea jecorina.

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.

Identification of a digalactosyl ononitol from seeds of adzuki bean (Vigna angularis).

A digalactosyl ononitol was isolated from seeds of adzuki bean (Vigna angularis [Willd.] Ohwi et Ohasi). Analysis of hydrolysis products and NMR spectroscopy established its structure as O-alpha-D-galactopyranosyl-(1-->6)-O-alpha-D-galactopyranosyl-(1-->3)-4-O-methyl-D-myo-inositol.

Functional expression of a cDNA encoding pea (Pisum sativum L.) raffinose synthase, partial purification of the enzyme from maturing seeds, and steady-state kinetic analysis of raffinose synthesis.

Raffinose (O-alpha- D-galactopyranosyl-(1-->6)- O-alpha- D-glucopyranosyl-(1<-->2)- O-beta- D-fructofuranoside) is a widespread oligosaccharide in plant seeds and other tissues. Raffinose synthase (EC 2.4.1.82) is the key enzyme that channels sucrose into the raffinose oligosaccharide pathway. We here report on the isolation of a cDNA encoding for raffinose synthase from maturing pea ( Pisum sativum L.) seeds. The coding region of the cDNA was expressed in Spodoptera frugiperda Sf21 insect cells. The recombinant enzyme, a protein of glycoside hydrolase family 36, displayed similar kinetic properties to raffinose synthase partially purified from maturing seeds by anion-exchange and size-exclusion chromatography. Apart from the natural galactosyl donor galactinol ( O-alpha- D-galactopyranosyl-(1-->1)- L- myo-inositol), p-nitrophenyl alpha- D-galactopyranoside, an artificial substrate, was utilized as a galactosyl donor. An equilibrium constant of 4.1 was determined for the galactosyl transfer reaction from galactinol to sucrose. Steady-state kinetic analysis suggested that raffinose synthase is a transglycosidase operating by a ping-pong reaction mechanism and may also act as a glycoside hydrolase. The enzyme was strongly inhibited by 1-deoxygalactonojirimycin, a potent inhibitor for alpha-galactosidases (EC 3.2.1.22). The physiological implications of these observations are discussed.

Chain Elongation of raffinose in pea seeds. Isolation, characterization, and molecular cloning of mutifunctional enzyme catalyzing the synthesis of stachyose and verbascose.

Raffinose oligosaccharides are major soluble carbohydrates in seeds and other tissues of plants. Their biosynthesis proceeds by stepwise addition of galactose units to sucrose, which are provided by the unusual donor galactinol (O-alpha-d-galactopyranosyl-(1-->1)-l-myo-inositol). Chain elongation may also proceed by transfer of galactose units between raffinose oligosaccharides. We here report on the purification, characterization, and heterologous expression of a multifunctional stachyose synthase (EC ) from developing pea (Pisum sativum L.) seeds. The protein, a member of family 36 of glycoside hydrolases, catalyzes the synthesis of stachyose, the tetrasaccharide of the raffinose series, by galactosyl transfer from galactinol to raffinose. It also mediates the synthesis of the pentasaccharide verbascose by galactosyl transfer from galactinol to stachyose as well as by self-transfer of the terminal galactose residue from one stachyose molecule to another. These activities show optima at pH 7.0. The enzyme also catalyzes hydrolysis of the terminal galactose residue of its substrates, but is unable to initiate the synthesis of raffinose oligosaccharides by galactosyl transfer from galactinol to sucrose. A minimum reaction mechanism which accounts for the broad substrate specificity and the steady-state kinetic properties of the protein is presented.