Huntington toxicity in yeast model depends on polyglutamine aggregation mediated by a prion-like protein Rnq1

The cause of Huntington's disease is expansion of polyglutamine (polyQ) domain in huntingtin, which makes this protein both neurotoxic and aggregation prone. Here we developed the first yeast model, which establishes a direct link between aggregation of expanded polyQ domain and its cytotoxicity. Our data indicated that deficiencies in molecular chaperones Sis1 and Hsp104 inhibited seeding of polyQ aggregates, whereas ssa1, ssa2, and ydj1-151 mutations inhibited expansion of aggregates. The latter three mutants strongly suppressed the polyQ toxicity. Spontaneous mutants with suppressed aggregation appeared with high frequency, and in all of them the toxicity was relieved. Aggregation defects in these mutants and in sis1-85 were not complemented in the cross to the hsp104 mutant, demonstrating an unusual type of inheritance. Since Hsp104 is required for prion maintenance in yeast, this suggested a role for prions in polyQ aggregation and toxicity. We screened a set of deletions of nonessential genes coding for known prions and related proteins and found that deletion of the RNQ1 gene specifically suppressed aggregation and toxicity of polyQ. Curing of the prion form of Rnq1 from wild-type cells dramatically suppressed both aggregation and toxicity of polyQ. We concluded that aggregation of polyQ is critical for its toxicity and that Rnq1 in its prion conformation plays an essential role in polyQ aggregation leading to the toxicity.     

Codependent functions of RSK2 and the apoptosis-promoting factor TIA-1 in stress granule assembly and cell survival

Stress granules aid cell survival in response to environmental stressors by acting as sites of translational repression. We report an unanticipated link between stress granules and the serine/threonine kinase RSK2. In stressed breast cells, endogenous RSK2 colocalizes in granules with TIA-1 and poly(A)-binding protein 1, and the sequestration of RSK2 and TIA-1 exhibits codependency. The RSK2 N-terminal kinase domain controls the direct interaction with the prion-related domain of TIA-1. Silencing RSK2 decreases cell survival in response to stress. Mitogen releases RSK2 from the stress granules and permits its nuclear import via a nucleocytoplasmic shuttling sequence in the C-terminal domain. Nuclear accumulation is dependent on TIA-1. Surprisingly, nuclear localization of RSK2 is sufficient to enhance proliferation through induction of cyclin D1, in the absence of other active signaling pathways. Hence, RSK2 is a pivotal factor linking the stress response to survival and proliferation.     

Human mitochondrial complex I assembly is mediated by NDUFAF1

Complex I (NADH:ubiquinone oxidoreductase) is the largest multiprotein enzyme of the oxidative phosphorylation system. Its assembly in human cells is poorly understood and no proteins assisting this process have yet been described. A good candidate is NDUFAF1, the human homologue of Neurospora crassa complex I chaperone CIA30. Here, we demonstrate that NDUFAF1 is a mitochondrial protein that is involved in the complex I assembly process. Modulating the intramitochondrial amount of NDUFAF1 by knocking down its expression using RNA interference leads to a reduced amount and activity of complex I. NDUFAF1 is associated to two complexes of 600 and 700 kDa in size of which the relative distribution is altered in two complex I deficient patients. Analysis of NDUFAF1 expression in a conditional complex I assembly system shows that the 700 kDa complex may represent a key step in the complex I assembly process. Based on these data, we propose that NDUFAF1 is an important protein for the assembly/stability of complex I.     

Junctional actin assembly is mediated by Formin-like 2 downstream of Rac1

Epithelial integrity is vitally important, and its deregulation causes early stage cancer. De novo formation of an adherens junction (AJ) between single epithelial cells requires coordinated, spatial actin dynamics, but the mechanisms steering nascent actin polymerization for cell–cell adhesion initiation are not well understood. Here we investigated real-time actin assembly during daughter cell–cell adhesion formation in human breast epithelial cells in 3D environments. We identify formin-like 2 (FMNL2) as being specifically required for actin assembly and turnover at newly formed cell–cell contacts as well as for human epithelial lumen formation. FMNL2 associates with components of the AJ complex involving Rac1 activity and the FMNL2 C terminus. Optogenetic control of Rac1 in living cells rapidly drove FMNL2 to epithelial cell–cell contact zones. Furthermore, Rac1-induced actin assembly and subsequent AJ formation critically depends on FMNL2. These data uncover FMNL2 as a driver for human epithelial AJ formation downstream of Rac1.     

The formation of Escherichia coli curli amyloid fibrils is mediated by prion-like peptide repeats

Amyloid fibril formation is the hallmark of major human maladies including Alzheimer's disease, type II diabetes, and prion diseases. Prion-like phenomena were also observed in yeast. Although not evolutionarily related, one similarity between the animal PrP and the yeast Sup35 prion proteins is the occurrence of short peptide repeats that are assumed to play a key role in the assembly of the amyloid structures. It was recently demonstrated that typical amyloid fibril formation is associated with biofilm formation by Escherichia coli. Here, we note the functional and structural similarity between oligopeptide repeats of the major curli protein and those of animal and yeast prions. We demonstrate that synthetic peptides corresponding to the repeats form fibrillar structures. Furthermore, conjugation of beta-breaker elements to the prion-like repeat significantly inhibits amyloid formation and cell invasion of curli-expressing bacteria. This implies a functional role of the repeat in the self-assembly of the fibrils. Since mammal prion, yeast prion, and curli protein are evolutionarily distinct, the conserved peptide repeats most likely define an optimized self-association motif that was independently evolved by diverse systems.     

Manipulating the aggregation activity of human prion-like proteins

Considerable advances in understanding the protein features favoring prion formation in yeast have facilitated the development of effective yeast prion prediction algorithms. Here we discuss a recent study in which we systematically explored the utility of the yeast prion prediction algorithm PAPA for designing mutations to modulate the aggregation activity of the human prion-like protein hnRNPA2B1. Mutations in hnRNPA2B1 cause multisystem proteinopathy in humans, and accelerate aggregation of the protein in vitro. Additionally, mutant hnRNPA2B1 forms cytoplasmic inclusions when expressed in Drosophila, and the mutant prion-like domain can substitute for a portion of a yeast prion domain in supporting prion activity in yeast. PAPA was quite successful at predicting the effects of PrLD mutations on prion activity in yeast and on in vitro aggregation propensity. Additionally, PAPA successfully predicted the effects of most, but not all, mutations in the PrLD of the hnRNPA2B1 protein when expressed in Drosophila. These results suggest that PAPA is quite effective at predicting the effects of mutations on intrinsic aggregation propensity, but that intracellular factors can influence aggregation and prion-like activity in vivo. A more complete understanding of these intracellular factors may inform the next generation of prion prediction algorithms.     

Filamin is essential in actin cytoskeletal assembly mediated by p21-activated kinase 1

The serine/threonine kinase p21-activated kinase 1 (Pak1) controls the actin cytoskeletal and ruffle formation through mechanisms that are independent of GTPase activity. Here we identify filamin FLNa as a Pak1-interacting protein through a yeast two-hybrid screen using the amino terminus of Pak1 as a bait. FLNa is stimulated by physiological signalling molecules to undergo phosphorylation by Pak1 and to interact and colocalize with endogenous Pak1 in membrane ruffles. The ruffle-forming activity of Pak1 is functional in FLNa-expressing cells but not in FLNa-deficient cells. In FLNa, the Pak1-binding site involves tandem repeat 23 in the carboxyl terminus and phosphorylation takes place on serine 2152. The FLNa-binding site in Pak1 is localized between amino acids 52 and 132 in the conserved Cdc42/Rac-interacting (CRIB) domain; accordingly, FLNa binding to the CRIB domain stimulates Pak1 kinase activity. Our results indicate that FLNa may be essential for Pak1-induced cytoskeletal reorganization and that the two-way regulatory interaction between Pak1 and FLNa may contribute to the local stimulation of Pak1 activity and its targets in cytoskeletal structures.     

Actin microfilament aggregation induced by withaferin A is mediated by annexin II

The actin cytoskeleton supports diverse cellular processes such as endocytosis, oriented growth, adhesion and migration. The dynamic nature of the cytoskeleton, however, has made it difficult to define the roles of the many accessory molecules that modulate actin organization, especially the multifunctional adapter protein annexin II. We now report that the compound withaferin A (1) can alter cytoskeletal architecture in a previously unknown manner by covalently binding annexin II and stimulating its basal F-actin cross-linking activity. Drug-mediated disruption of F-actin organization is dependent on annexin II expression by cells and markedly limits their migratory and invasive capabilities at subcytotoxic concentrations. Given the extensive ethnobotanical history of withaferin-containing plant preparations in the treatment of cancer and inflammatory and neurological disorders, we suggest that annexin II represents a feasible, previously unexploited target for therapeutic intervention by small-molecule drugs.     

Plastid division is mediated by combinatorial assembly of plastid division proteins

Plastids arise by division from pre-existing organelles, and with the recent characterization of several new components of plastid division our understanding of the division process in higher plants has improved dramatically. However, it is still not known how these different protein components act together during division. Here we analyse protein-protein interactions between all known stromal plastid division proteins. Using a combination of quantitative yeast two-hybrid assays, in planta co-localization studies, fluorescence resonance energy transfer and bimolecular fluorescence complementation assays we show that these proteins do not act in isolation but rather in protein complexes to govern appropriate plastid division. We have previously shown that AtMinD1 forms functional homodimers and we show here that in addition to homodimerization AtMinD1 also interacts with AtMinE1. Furthermore, AtMinE1 has the ability to homodimerize. We also demonstrate that proteins from both FtsZ families (AtFtsZ1-1 and AtFtsZ2-1) not only interact with themselves but also with each other, and we show that these interactions are not dependent on correct Z-ring formation. Further to this we demonstrate that ARC6 specifically interacts with the core domain of AtFtsZ2-1, but not with AtFtsZ1-1, providing in planta evidence for a functional difference between the two FtsZ protein families in plants. Our studies have enabled us to construct a meaningful intraplastidic protein-protein interaction map of all known stromal plastid division proteins in Arabidopsis.     

Perylene diimide G-quadruplex DNA binding selectivity is mediated by ligand aggregation

Two N,N'-disubstituted perylene diimide G-quadruplex DNA ligands, PIPER [N,N'-bis-(2-(1-piperidino)ethyl)-3,4,9,10-perylene tetracarboxylic acid diimide] and Tel01 [N,N'-bis-(3-(4-morpholino)-propyl)-3,4,9,10-perylene tetracarboxylic acid diimide] were studied. Visible absorbance, resonance light scattering, and fluorescence spectroscopy were used to characterize the pH-dependent aggregation of these ligands. The G-quadruplex DNA binding selectivity of these ligands as monitored by absorption spectroscopy is also pH-dependent. The ligands bind to both duplex and G-quadruplex DNA under low pH conditions, where the ligands are not aggregated. At higher pH, where the ligands are extensively aggregated, the apparent G-quadruplex DNA binding selectivity is high.     

The aggregation of mutant p53 produces prion-like properties in cancer

The tumor suppressor protein p53 loses its function in more than 50% of human malignant tumors. Recent studies have suggested that mutant p53 can form aggregates that are related to loss-of-function effects, negative dominance and gain-of-function effects and cancers with a worsened prognosis. In recent years, several degenerative diseases have been shown to have prion-like properties similar to mammalian prion proteins (PrPs). However, whereas prion diseases are rare, the incidence of these neurodegenerative pathologies is high. Malignant tumors involving mutated forms of the tumor suppressor p53 protein seem to have similar substrata. The aggregation of the entire p53 protein and three functional domains of p53 into amyloid oligomers and fibrils has been demonstrated. Amyloid aggregates of mutant p53 have been detected in breast cancer and malignant skin tumors. Most p53 mutations related to cancer development are found in the DNA-binding domain (p53C), which has been experimentally shown to form amyloid oligomers and fibrils. Several computation programs have corroborated the predicted propensity of p53C to form aggregates, and some of these programs suggest that p53C is more likely to form aggregates than the globular domain of PrP. Overall, studies imply that mutant p53 exerts a dominant-negative regulatory effect on wild-type (WT) p53 and exerts gain-of-function effects when co-aggregating with other proteins such as p63, p73 and acetyltransferase p300. We review here the prion-like behavior of oncogenic p53 mutants that provides an explanation for their dominant-negative and gain-of-function properties and for the high metastatic potential of cancers bearing p53 mutations. The inhibition of the aggregation of p53 into oligomeric and fibrillar amyloids appears to be a promising target for therapeutic intervention in malignant tumor diseases.     

Two isoforms of the T-cell intracellular antigen 1 (TIA-1) splicing factor display distinct splicing regulation activities. Control of TIA-1 isoform ratio by TIA-1-related protein

TIA-1 (T-cell Intracellular Antigen 1) and TIAR (TIA-1-related protein) are RNA-binding proteins involved in the regulation of alternative pre-mRNA splicing and other aspects of RNA metabolism. Various isoforms of these proteins exist in mammals. For example, TIA-1 presents two major isoforms (TIA-1a and TIA-1b) generated by alternative splicing of exon 5 that differ by eleven amino acids exclusive of the TIA-1a isoform. Here we show that the relative expression of TIA-1 and TIAR isoforms varies in different human tissues and cell lines, suggesting distinct functional properties and regulated isoform expression. We report that whereas TIA-1 isoforms show similar subcellular distribution and RNA binding, TIA-1b displays enhanced splicing stimulatory activity compared with TIA-1a, both in vitro and in vivo. Interestingly, TIAR depletion from HeLa and mouse embryonic fibroblasts results in an increased ratio of TIA-1b/a expression, suggesting that TIAR regulates the relative expression of TIA-1 isoforms. Taken together, the results reveal distinct functional properties of TIA-1 isoforms and the existence of a regulatory network that controls isoform expression.     

Hemicentin assembly in the extracellular matrix is mediated by distinct structural modules

Hemicentins are conserved extracellular matrix proteins characterized by a single von Willebrand A (VWA) domain at the amino terminus, a long stretch (>40) of tandem immunoglobulin domains, multiple tandem epidermal growth factors (EGFs), and a single fibulin-like carboxyl-terminal module. In Caenorhabditis elegans, hemicentin is secreted from muscle and gonadal leader cells and assembles at multiple locations into discrete tracks that constrict broad regions of cell contact into adhesive and flexible line-shaped junctions. To determine hemicentin domains critical for function and assembly, we have expressed fragments of hemicentin as GFP tagged fusion proteins in C. elegans. We find that a hemicentin fragment containing the VWA domain can target to multiple assembly sites when expressed under the control of either endogenous hemicentin regulatory sequences or the muscle-specific unc-54 promoter. A hemicentin fragment containing the EGF and fibulin-like carboxyl-terminal modules can co-assemble with existing hemicentin polymers in wild-type animals but has no detectable function in the absence of endogenous hemicentin. The data suggest that the VWA domain is a cell binding domain whose function is to target hemicentin to sites of assembly and the EGF/fibulin-like carboxyl-terminal modules constitute an assembly domain that mediates direct interactions between hemicentin monomers during the hemicentin assembly process.     

The apoptosis-promoting factor TIA-1 is a regulator of alternative pre-mRNA splicing

We report here that the apoptosis-promoting protein TIA-1 regulates alternative pre-mRNA splicing of the Drosophila melanogaster gene male-specific-lethal 2 and of the human apoptotic gene Fas. TIA-1 associates selectively with pre-mRNAs that contain 5' splice sites followed by U-rich sequences. TIA-1 binding to the U-rich stretches facilitates 5' splice site recognition by U1 snRNP. This activity is critical for activation of the weak 5' splice site of msl-2 and for modulating the choice of splice site partner in Fas. Structural and functional similarities with the Saccharomyces cerevisiae splicing factor Nam8 suggest striking evolutionary conservation of a mechanism of pre-mRNA splicing regulation that controls biological processes as diverse as meiosis in yeast, dosage compensation in fruit flies, or programmed cell death in humans.     

Poliovirus unlinks TIA1 aggregation and mRNA stress granule formation

In response to environmental stress and viral infection, mammalian cells form foci containing translationally silenced mRNPs termed stress granules (SGs). As aggregates of stalled initiation complexes, SGs are defined by the presence of translation initiation machinery in addition to mRNA binding proteins. Here, we report that cells infected with poliovirus (PV) can form SGs early that contain T-cell-restricted intracellular antigen 1 (TIA1), translation initiation factors, RNA binding proteins, and mRNA. However, this response is blocked as infection progresses, and a type of pseudo-stress granule remains at late times postinfection and contains TIA but lacks translation initiation factors, mRNA binding proteins, and most polyadenylated mRNA. This result was observed using multiple stressors, including viral infection, oxidative stress, heat shock, and endoplasmic reticulum stress. Multiple proteins required for efficient viral internal ribosome entry site-dependent translation are localized to SGs under stress conditions, providing a potential rationale for the evolution and maintenance of the SG inhibition phenotype. Further, the expression of a noncleavable form of the RasGAP-SH3 domain binding protein in PV-infected cells enables SGs whose constituents are consistent with the presence of stalled 48S translation preinitiation complexes to persist throughout infection. These results indicate that in poliovirus-infected cells, the functions of TIA self-aggregation and aggregation of stalled translation initiation complexes into stress granules are severed, leading to novel foci that contain TIA1 but lack other stress granule-defining components.     

Inhibitors of amyloid beta-protein aggregation mediated by GM1-containing raft-like membranes

The aggregation (fibril formation) of amyloid beta-protein (Abeta) is considered to be a crucial step in the etiology of Alzheimer's disease (AD). The inhibition of Abeta aggregation and/or decomposition of fibrils formed in aqueous solution by small compounds have been studied extensively for the prevention and treatment of AD. However, recent studies suggest that Abeta aggregation also occurs in lipid rafts mediated by a cluster of monosialoganglioside GM1. This study examined the effects of representative compounds on Abeta aggregation and fibril destabilization in the presence of GM1-containing raft-like liposomes. Among the compounds tested, nordihydroguaiaretic acid (NDGA), rifampicin (RIF), tannic acid (TA), and quercetin (QUE) showed strong fibrillization inhibitory activity. NDGA and RIF inhibited the binding of Abeta to GM1 liposomes by competitively binding to the membranes and/or direct interaction with Abeta in solution, thus at least partly preventing fibrils from forming. Coincubation of Abeta with NDGA, RIF, and QUE in the presence of GM1 liposomes resulted in elongate particles, whereas the presence of TA yielded protofibrillar structures. TA and RIF also destabilized fibrils. The most potent NDGA prevented Abeta-induced toxicity in PC12 cells by inhibiting Abeta accumulation. Furthermore, a comparison of the inhibitory effects of various compounds between aqueous-phase and GM1-mediated aggregation of Abeta suggested that the two aggregation processes are not identical.     

Secretagogue-dependent phosphorylation of the insulin granule membrane protein phogrin is mediated by cAMP-dependent protein kinase

Phogrin, a 60/64-kDa integral membrane protein of dense-core granules in neuroendocrine cells, is phosphorylated in a Ca(2+)-sensitive manner in response to secretagogue stimulation of pancreatic beta-cells. Phosphorylation of the phogrin cytosolic domain by beta-cell homogenates was Ca(2+)-independent but stimulated by cAMP. Recombinant protein kinase A (PKA) could phosphorylate phogrin directly. High performance liquid chromatography analysis of tryptic phosphopeptides, combined with site-directed mutagenesis of candidate sites, revealed the presence of two phosphorylation sites at Ser-680 and Thr-699, located in the juxtamembrane region between the transmembrane span and the protein-tyrosine phosphatase homology domain of phogrin. Full-length wild-type phogrin, as well as mutant versions where Ser-680 and Thr-699 had been replaced either by alanines or by aspartic acid residues, were targeted to secretory granules in transfected AtT20 neuroendocrine cells. Stimulation of these cells with a range of secretagogues, including K(+), BaCl(2), and forskolin, demonstrated that the in vivo phosphorylation sites are the same as those identified in vitro. In MIN6 beta-cells, the PKA inhibitor H-89 prevented Ca(2+)-dependent phogrin phosphorylation in response to glucose, suggesting that Ca(2+) exerts its effect on phogrin phosphorylation through regulating the activity of PKA.