Phase-separated ribosome-nascent chain complexes in genotoxic stress response.

Assemblysomes are EDTA- and RNase-resistant ribonucleoprotein (RNP) complexes of paused ribosomes with protruding nascent polypeptide chains. They have been described in yeast and human cells for the proteasome subunit Rpt1, and the disordered amino-terminal part of the nascent chain was found to be indispensable for the accumulation of the Rpt1-RNP into assemblysomes. Motivated by this, to find other assemblysome-associated RNPs we used bioinformatics to rank subunits of Saccharomyces cerevisiae protein complexes according to their amino-terminal disorder propensity. The results revealed that gene products involved in DNA repair are enriched among the top candidates. The Sgs1 DNA helicase was chosen for experimental validation. We found that indeed nascent chains of Sgs1 form EDTA-resistant RNP condensates, assemblysomes by definition. Moreover, upon exposure to UV, SGS1 mRNA shifted from assemblysomes to polysomes, suggesting that external stimuli are regulators of assemblysome dynamics. We extended our studies to human cell lines. The BLM helicase, ortholog of yeast Sgs1, was identified upon sequencing assemblysome-associated RNAs from the MCF7 human breast cancer cell line, and mRNAs encoding DNA repair proteins were overall enriched. Using the radiation-resistant A549 cell line, we observed by transmission electron microscopy that 1,6-hexanediol, an agent known to disrupt phase-separated condensates, depletes ring ribosome structures compatible with assemblysomes from the cytoplasm of cells and makes the cells more sensitive to X-ray treatment. Taken together, these findings suggest that assemblysomes may be a component of the DNA damage response from yeast to human.

Interaction of the sorting nexin 25 homologue Snazarus with Rab11 balances endocytic and secretory transport and maintains the ultrafiltration diaphragm in nephrocytes.

Proper balance of exocytosis and endocytosis is important for the maintenance of plasma membrane lipid and protein homeostasis. This is especially critical in human podocytes and the podocyte-like Drosophila nephrocytes that both use a delicate diaphragm system with evolutionarily conserved components for ultrafiltration. Here, we show that the sorting nexin 25 homologue Snazarus (Snz) binds to Rab11 and localizes to Rab11-positive recycling endosomes in Drosophila nephrocytes, unlike in fat cells where it is present in plasma membrane/lipid droplet/endoplasmic reticulum contact sites. Loss of Snz leads to redistribution of Rab11 vesicles from the cell periphery and increases endocytic activity in nephrocytes. These changes are accompanied by defects in diaphragm protein distribution that resemble those seen in Rab11 gain-of-function cells. Of note, co-overexpression of Snz rescues diaphragm defects in Rab11 overexpressing cells, whereas snz knockdown in Rab11 overexpressing nephrocytes or simultaneous knockdown of snz and tbc1d8b encoding a Rab11 GTPase-activating protein (GAP) leads to massive expansion of the lacunar system that contains mislocalized diaphragm components: Sns and Pyd/ZO-1. We find that loss of Snz enhances while its overexpression impairs secretion, which, together with genetic epistasis analyses, suggest that Snz counteracts Rab11 to maintain the diaphragm via setting the proper balance of exocytosis and endocytosis.

Reversible binding of divalent cations to Ductin protein assemblies-A putative new regulatory mechanism of membrane traffic processes.

Ductins are a family of homologous and structurally similar membrane proteins with 2 or 4 trans-membrane alpha-helices. The active forms of the Ductins are membranous ring- or star-shaped oligomeric assemblies and they provide various pore, channel, gap-junction functions, assist in membrane fusion processes and also serve as the rotor c-ring domain of V-and F-ATPases. All functions of the Ductins have been reported to be sensitive to the presence of certain divalent metal cations (Me2+), most frequently Cu2+ or Ca2+ ions, for most of the better known members of the family, and the mechanism of this effect is not yet known. Given that we have earlier found a prominent Me2+ binding site in a well-characterised Ductin protein, we hypothesise that certain divalent cations can structurally modulate the various functions of Ductin assemblies via affecting their stability by reversible non-covalent binding to them. A fine control of the stability of the assembly ranging from separated monomers through a loosely/weakly to tightly/strongly assembled ring might render precise regulation of Ductin functions possible. The putative role of direct binding of Me2+ to the c-ring subunit of active ATP hydrolase in autophagy and the mechanism of Ca2+-dependent formation of the mitochondrial permeability transition pore are also discussed.

Prolonged activity of the transposase helper may raise safety concerns during DNA transposon-based gene therapy.

DNA transposon-based gene delivery vectors represent a promising new branch of randomly integrating vector development for gene therapy. For the side-by-side evaluation of the piggyBac and Sleeping Beauty systems-the only DNA transposons currently employed in clinical trials-during therapeutic intervention, we treated the mouse model of tyrosinemia type I with liver-targeted gene delivery using both transposon vectors. For genome-wide mapping of transposon insertion sites we developed a new next-generation sequencing procedure called streptavidin-based enrichment sequencing, which allowed us to identify approximately one million integration sites for both systems. We revealed that a high proportion of piggyBac integrations are clustered in hot regions and found that they are frequently recurring at the same genomic positions among treated animals, indicating that the genome-wide distribution of Sleeping Beauty-generated integrations is closer to random. We also revealed that the piggyBac transposase protein exhibits prolonged activity, which predicts the risk of oncogenesis by generating chromosomal double-strand breaks. Safety concerns associated with prolonged transpositional activity draw attention to the importance of squeezing the active state of the transposase enzymes into a narrower time window.

Ion Channels and Pumps in Autophagy: A Reciprocal Relationship.

Autophagy, the process of cellular self-degradation, is intrinsically tied to the degradative function of the lysosome. Several diseases have been linked to lysosomal degradative defects, including rare lysosomal storage disorders and neurodegenerative diseases. Ion channels and pumps play a major regulatory role in autophagy. Importantly, calcium signaling produced by TRPML1 (transient receptor potential cation channel, mucolipin subfamily) has been shown to regulate autophagic progression through biogenesis of autophagic-lysosomal organelles, activation of mTORC1 (mechanistic target of rapamycin complex 1) and degradation of autophagic cargo. ER calcium channels such as IP3Rs supply calcium for the lysosome, and lysosomal function is severely disrupted in the absence of lysosomal calcium replenishment by the ER. TRPML1 function is also regulated by LC3 (microtubule-associated protein light chain 3) and mTORC1, two critical components of the autophagic network. Here we provide an overview of the current knowledge about ion channels and pumps-including lysosomal V-ATPase (vacuolar proton-ATPase), which is required for acidification and hence proper enzymatic activity of lysosomal hydrolases-in the regulation of autophagy, and discuss how functional impairment of some of these leads to diseases.

Combination of Alanine and Glutathione as Targeting Ligands of Nanoparticles Enhances Cargo Delivery into the Cells of the Neurovascular Unit.

Inefficient drug delivery across the blood-brain barrier (BBB) and into target cells in the brain hinders the treatment of neurological diseases. One strategy to increase the brain penetration of drugs is to use vesicular nanoparticles functionalized with multiple ligands of BBB transporters as vehicles. Once within the brain, however, drugs must also be able to reach their therapeutic targets in the different cell types. It is, therefore, favorable if such nanocarriers are designed that can deliver their cargo not only to brain endothelial cells, but to other cell types as well. Here, we show that alanineglutathione dual-targeting of niosomes enhances the delivery of a large protein cargo into cultured cells of the neurovascular unit, namely brain endothelial cells, pericytes, astrocytes and neurons. Furthermore, using metabolic and endocytic inhibitors, we show that the cellular uptake of niosomes is energy-dependent and is partially mediated by endocytosis. Finally, we demonstate the ability of our targeted nanovesicles to deliver their cargo into astroglial cells after crossing the BBB in vitro. These data indicate that dual-labeling of nanoparticles with alanine and glutathione can potentially be exploited to deliver drugs, even biopharmacons, across the BBB and into multiple cell types in the brain.

Cross-Species Interaction between Rapidly Evolving Telomere-Specific Drosophila Proteins.

Telomere integrity in Drosophila melanogaster is maintained by a putative multisubunit complex called terminin that is believed to act in analogy to the mammalian shelterin complex in protecting chromosome ends from being recognized as sites of DNA damage. The five proteins supposed to form the terminin complex are HP1-ORC associated protein, HP1-HOAP interacting protein, Verrocchio, Drosophila Telomere Loss/Modigliani and Heterochromatic Protein 1. Four of these proteins evolve rapidly within the Drosophila genus. The accelerated evolution of terminin components may indicate the involvement of these proteins in the process by which new species arise, as the resulting divergence of terminin proteins might prevent hybrid formation, thus driving speciation. However, terminin is not an experimentally proven entity, and no biochemical studies have been performed to investigate its assembly and action in detail. Motivated by these facts in order to initiate biochemical studies on terminin function, we attempted to reconstitute terminin by co-expressing its subunits in bacteria and investigated the possible role of the fast-evolving parts of terminin components in complex assembly. Our results suggest formation of stable subcomplexes of terminin, but not of the whole complex in vitro. We found that the accelerated evolution is restricted to definable regions of terminin components, and that the divergence of D. melanogaster Drosophila Telomere Loss and D. yakuba Verrocchio proteins does not preclude their stable interaction.

DNA replication: damage tolerance at the assembly line.

Damage tolerance mechanisms ensure resumption of DNA synthesis at damage-replisome encounters. Replication fork reversal (RFR) is one such widely recognized mechanism that acts on replisomes where lagging strand synthesis continues upon leading strand synthesis block. The possibility to form such a structure is highly counter to our current understanding of the replisome dynamics of single replisomes. Here, I suggest a model that takes coupled bidirectional replisome organization into account to solve this apparent contradiction.

Functional analysis of long-chain n-alkane degradation by Dietzia spp.

The genetic background of long-chain n-alkane degradation was investigated in detail in strain E1, a member of the genetically unexplored Dietzia genus. A suicide vector carrying a 518-bp alkB fragment was site-specifically integrated into the E1 chromosome, and the full alkB, as well as its chromosomal environment was sequenced after plasmid rescue experiments. Four out of the nine putative genes were strongly induced by long-chain n-alkanes in wild-type E1. ORF4 encoded a natural fusion protein consisting of an integral membrane alkane hydroxylase and a rubredoxin domain. The significance of the alkB-rub gene in n-alkane degradation was investigated in phenotypic tests, and the disruption mutant strain exhibited severely impaired growth on n-C(20) alkane carbon source. The mutation was successfully complemented with the expression of intact AlkB-Rub protein, the full-length form of which was detected by simultaneous immunoblotting. The presented data furnish the first experimental evidence of the in vivo existence of an AlkB-Rub natural fusion protein, which plays a major role in long-chain n-alkane degradation.

Role of yeast Rad5 and its human orthologs, HLTF and SHPRH in DNA damage tolerance.

In the yeast Saccharomyces cerevisiae, the Rad6-Rad18 DNA damage tolerance pathway constitutes a major defense system against replication fork blocking DNA lesions. The Rad6-Rad18 ubiquitin-conjugating/ligase complex governs error-free and error-prone translesion synthesis by specialized DNA polymerases, as well as an error-free Rad5-dependent postreplicative repair pathway. For facilitating replication through DNA lesions, translesion synthesis polymerases copy directly from the damaged template, while the Rad5-dependent damage tolerance pathway obtains information from the newly synthesized strand of the undamaged sister duplex. Although genetic data demonstrate the importance of the Rad5-dependent pathway in tolerating DNA damages, there has been little understanding of its mechanism. Also, the conservation of the yeast Rad5-dependent pathway in higher order eukaryotic cells remained uncertain for a long time. Here we summarize findings published in recent years regarding the role of Rad5 in promoting error-free replication of damaged DNA, and we also discuss results obtained with its human orthologs, HLTF and SHPRH.

Role of double-stranded DNA translocase activity of human HLTF in replication of damaged DNA.

Unrepaired DNA lesions can block the progression of the replication fork, leading to genomic instability and cancer in higher-order eukaryotes. In Saccharomyces cerevisiae, replication through DNA lesions can be mediated by translesion synthesis DNA polymerases, leading to error-free or error-prone damage bypass, or by Rad5-mediated template switching to the sister chromatid that is inherently error free. While translesion synthesis pathways are highly conserved from yeast to humans, very little is known of a Rad5-like pathway in human cells. Here we show that a human homologue of Rad5, HLTF, can facilitate fork regression and has a role in replication of damaged DNA. We found that HLTF is able to reverse model replication forks, a process which depends on its double-stranded DNA translocase activity. Furthermore, from analysis of isolated dually labeled chromosomal fibers, we demonstrate that in vivo, HLTF promotes the restart of replication forks blocked at DNA lesions. These findings suggest that HLTF can promote error-free replication of damaged DNA and support a role for HLTF in preventing mutagenesis and carcinogenesis, providing thereby for its potential tumor suppressor role.

The transcriptional coactivator SAYP is a trithorax group signature subunit of the PBAP chromatin remodeling complex.

SWI/SNF ATP-dependent chromatin remodeling complexes (remodelers) perform critical functions in eukaryotic gene expression control. BAP and PBAP are the fly representatives of the two evolutionarily conserved major subclasses of SWI/SNF remodelers. Both complexes share seven core subunits, including the Brahma ATPase, but differ in a few signature subunits; POLYBROMO and BAP170 specify PBAP, whereas OSA defines BAP. Here, we show that the transcriptional coactivator and PHD finger protein SAYP is a novel PBAP subunit. Biochemical analysis established that SAYP is tightly associated with PBAP but absent from BAP. SAYP, POLYBROMO, and BAP170 display an intimately overlapping distribution on larval salivary gland polytene chromosomes. Genome-wide expression analysis revealed that SAYP is critical for PBAP-dependent transcription. SAYP is required for normal development and interacts genetically with core- and PBAP-selective subunits. Genetic analysis suggested that, like BAP, PBAP also counteracts Polycomb silencing. SAYP appears to be a key architectural component required for the integrity and association of the PBAP-specific module. We conclude that SAYP is a signature subunit that plays a major role in the functional specificity of the PBAP holoenzyme.

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

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.

Human SHPRH is a ubiquitin ligase for Mms2-Ubc13-dependent polyubiquitylation of proliferating cell nuclear antigen.

Human SHPRH gene is located at the 6q24 chromosomal region, and loss of heterozygosity in this region is seen in a wide variety of cancers. SHPRH is a member of the SWI/SNF family of ATPases/helicases, and it possesses a C(3)HC(4) RING motif characteristic of ubiquitin ligase proteins. In both of these features, SHPRH resembles the yeast Rad5 protein, which, together with Mms2-Ubc13, promotes replication through DNA lesions via an error-free postreplicational repair pathway. Genetic evidence in yeast has indicated a role for Rad5 as a ubiquitin ligase in mediating the Mms2-Ubc13-dependent polyubiquitylation of proliferating cell nuclear antigen. Here we show that SHPRH is a functional homolog of Rad5. Similar to Rad5, SHPRH physically interacts with the Rad6-Rad18 and Mms2-Ubc13 complexes, and we show that SHPRH protein is a ubiquitin ligase indispensable for Mms2-Ubc13-dependent polyubiquitylation of proliferating cell nuclear antigen. Based on these observations, we predict a role for SHPRH in promoting error-free replication through DNA lesions. Such a role for SHPRH is consistent with the observation that this gene is mutated in a number of cancer cell lines, including those from melanomas and ovarian cancers, which raises the strong possibility that SHPRH function is an important deterrent to mutagenesis and carcinogenesis in humans.

Activation of an alfalfa cyclin-dependent kinase inhibitor by calmodulin-like domain protein kinase.

Kip-related proteins (KRPs) play a central role in the regulation of the cell cycle and differentiation through modulation of cyclin-dependent kinase (CDK) functions. We have identified a CDK inhibitor gene from Medicago truncatula (Mt) by a yeast two-hybrid screen. The KRPMt gene was expressed in all plant organs and cultured cells, and its transcripts accumulated after abscisic acid and NaCl treatment. The KRPMt protein exhibits seven conserved sequence domains and a PEST motif that is also detected in various Arabidopsis KRPs. In the yeast two-hybrid test, the KRPMt protein interacted with CDK (Medsa;CDKA;1) and D-type cyclins. However, in the pull-down assays, B-type CDK complexes were also detectable. Recombinant KRPMt differentially inhibited various alfalfa CDK complexes in phosphorylation assays. The immunoprecipitated Medsa;CDKA;1/A;2 complex was strongly inhibited, whereas the mitotic Medsa;CDKB2;1 complex was the most sensitive to inhibition. Function of Medsa;CDKB1;1 complex was not inhibited by the KRPMt protein. The mitotic Medsa;CYCB2 and Medsa;CYCA2;1 complexes responded weakly to this inhibitor protein. Kinase complexes from G2/M cells showed increased sensitivity towards the inhibitor compared with those isolated from G1/S-phase cells. In vitro phosphorylation of Medicago retinoblastoma-related protein was also reduced in the presence of KRPMt. Phosphorylation of this inhibitor protein by the recombinant calmodulin-like domain protein kinase (MsCPK3) resulted in enhanced inhibition of CDK function. The data presented emphasize the selective sensitivity of various cyclin-dependent kinase complexes to this inhibitor protein, and suggest a role for CDK inhibitors and CPKs in cross-talk between Ca2+ signalling and regulation of cell-cycle progression in plants.

Efficient and specific targeting of Polycomb group proteins requires cooperative interaction between Grainyhead and Pleiohomeotic.

Specific targeting of the protein complexes formed by the Polycomb group of proteins is critically required to maintain the inactive state of a group of developmentally regulated genes. Although the role of DNA binding proteins in this process has been well established, it is still not understood how these proteins target the Polycomb complexes specifically to their response elements. Here we show that the grainyhead gene, which encodes a DNA binding protein, interacts with one such Polycomb response element of the bithorax complex. Grainyhead binds to this element in vitro. Moreover, grainyhead interacts genetically with pleiohomeotic in a transgene-based, pairing-dependent silencing assay. Grainyhead also interacts with Pleiohomeotic in vitro, which facilitates the binding of both proteins to their respective target DNAs. Such interactions between two DNA binding proteins could provide the basis for the cooperative assembly of a nucleoprotein complex formed in vitro. Based on these results and the available data, we propose that the role of DNA binding proteins in Polycomb group-dependent silencing could be described by a model very similar to that of an enhanceosome, wherein the unique arrangement of protein-protein interaction modules exposed by the cooperatively interacting DNA binding proteins provides targeting specificity.