SIRT1 Deacetylase Activity and the Maintenance of Protein Homeostasis in Response to Stress: An Overview

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Coat protein and RNAs of Cole latent virus are not present within chloroplasts of Chenopodium quinoa-infected cells

O vírus latente da couve (Cole latent virus, CoLV), gênero Carlavirus, foi estudado, por microscopia eletrônica de transmissão e técnicas bioquímicas, em relação à ultra-estrutura das células infetadas de Chenopodium quinoa, e de sua associação com os cloroplastos. O CoLV foi observado como partículas dispersas pelo citoplasma entremeadas com vesículas membranosas e ribossomos e/ou como densas massas de partículas. Estes partículas reagiram por imunomarcação com anti-soro policlonal para o CoLV. Morfologicamente, cloroplastos, mitocôndrias e núcleos mostraram-se inalterados e partículas virais não foram encontradas dentro dessas organelas. Entretanto, agregados de partículas virais foram freqüentemente vistos em associação com a membrana externa dos cloroplastos e ocasionalmente com peroxissomos. Cloroplastos foram purificados em gradiente de Percoll e as proteínas e os RNA foram extraídos e analisados, respectivamente, por Western blot e Northern blot. Proteína capsidial e RNA associados ao CoLV não foram detectados nessa organela. Os resultados aqui obtidos indicam que a associação CoLV/cloroplastos, observada nos estudos de microscopia eletrônica, é possivelmente um evento casual dentro da célula hospedeira e que o vírus não se multiplica dentro dessa organela.

Crystal Structures of Xanthomonas Small Heat Shock Protein Provide a Structural Basis for an Active Molecular Chaperone Oligomer

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Human interferon B1 ser17: Coding DNA synthesis, expression, purification and characterization of bioactive recombinant protein

A protocol to produce large amounts of bioactive homogeneous human interferon β1 expressed in Escherichia coli was developed. Human interferon β1 ser17 gene was constructed, cloned and subcloned, and the recombinant protein expressed in E. coli cells. Solubilization of recombinant human interferon β1 ser17 (rhIFN-β1 ser17) was accomplished by employing a brief shift to high alkaline pH in the presence of non-ionic detergent. The recombinant protein was purifi ed by three chromatographic steps. N-terminal amino acid sequencing and mass spectrometry analysis provided experimental evidence for the identity of the recombinant protein. Reverse phase liquid chromatography demonstrated that the content of deamidates and sulphoxides was similar to a commercial standard. Size exclusion chromatography demonstrated the absence of high molecular mass aggregates and dimers. The protocol represents an effi cient and high-yield method to obtain bioactive homogeneous monomeric rhIFN-β1 ser17 protein. It may thus represent an important step towards scaling up for rhIFN-β1 ser17 large-scale production. © 2010 Villela AD, et al.

Accumulation of the SET protein in HEK293T cells and mild oxidative stress: cell survival or death signaling

SET protein (I2PP2A) is an inhibitor of PP2A, which regulates the phosphorylated Akt (protein kinase B) levels. We assessed the effects of SET overexpression in HEK293T cells, both in the presence and the absence of mild oxidative stress induced by 50 mu M tert-butyl hydroperoxide. Immunoblotting assays demonstrated that SET accumulated in HEK293T cells and increased the levels of phosphorylated Akt and PTEN; in addition, SET decreased glutathione antioxidant defense of cell and increased expression of genes encoding antioxidant defense proteins. Immunofluorescence analysis demonstrated that accumulated SET was equally distributed in cytoplasm and nucleus; however, in cells that had been exposed to oxidative stress, SET was found in large aggregates in the cytoplasm. SET accumulation in HEK293T cells correlated with inhibition of basal apoptosis as evidenced by a decrease in annexin V staining and activity of caspases; under mild oxidative stress, SET accumulation correlated with caspase-independent cell death, as evidenced by increased PI and annexin V/PI double staining. The results suggest that accumulated SET could act via Akt/PTEN either as cell survival signal or as oxidative stress sensor for cell death.

Genetic and physical analysis of the bacteriophage P22 tail protein

A plasmid based genetic system was developed for the tail protein of the Salmonella typhimurium bacteriophage P22 and used to isolate and characterize tail protein mutants. The tail protein is a trimeric structural protein of the phage and an endorhamnosidase whose activity is essential for infection. The gene for the tail protein has previously been cloned into a plasmid expression vector and sequenced. A plate complementation assay for tail protein produced from the cloned gene was developed and used to isolate 27 tail protein mutants following mutagenesis of the cloned gene. These mutations were mapped into 12 deletion intervals using deletions which were made on plasmids in vitro and crossed onto P22. The base substitutions were determined by DNA sequencing. The majority of mutants had missense or nonsense mutations in the protein coding portion of the gene; however four of the mutants were in the putative transcription terminator. The oligomeric state of tail protein from the 15 missense mutants was investigated using SDS and nondenaturing polyacrylamide gel electrophoresis of cell lysates. Wild-type tail protein retains its trimeric structure in SDS gels at room temperature. Two of the mutant proteins also migrated as trimers in SDS gels, yet one of these had a considerably faster mobility than wild-type trimer. Its migration was the same as wild-type in a nondenaturing gel, so it is thought to be a trimer which is partially denatured by SDS. Four of the mutants produced proteins which migrate at the position of a monomer in an SDS gel but cannot be seen on a nondenaturing gel. These proteins are thought to be either monomers or soluble aggregates which cannot enter the nondenaturing gel. The remainder of mutants produce protein which is degraded. The mutant tail protein which had normal trimeric mobility on SDS and nondenaturing gels was purified. This protein has essentially wild-type ability to attach to phage capsids, but its endorhamnosidase activity is only 4% of wild-type. ^

The multifunctional FUS protein: Characterization of the biological effects of its depletion

FUS/TLS (fused in sarcoma/translocated in liposarcoma) protein, a ubiquitously expressed RNA-binding protein, has been linked to a variety of cellular processes, such as RNA metabolism, microRNA biogenesis and DNA repair. However, the precise role of FUS protein remains unclear. Recently, FUS has been linked to Amyotrophic Lateral Sclerosis (ALS), a neurodegenerative disorder characterized by the dysfunction and death of motor neurons. Based on the observation that some mutations in the FUS gene induce cytoplasmic accumulation of FUS aggregates, we decided to explore a loss-of-function situation (i.e. inhibition of FUS’ nuclear function) to unravel the role of this protein. To this purpose, we have generated a SH-SY5Y human neuroblastoma cell line which expresses a doxycycline induced shRNA targeting FUS and that specifically depletes the protein. In order to characterize this cell line, we have performed a whole transcriptome analysis by RNA deep sequencing. Preliminary results show that FUS depletion affects both expression and alternative splicing levels of several RNAs. When FUS is depleted we observed 330 downregulated and 81 upregulated genes. We also found that 395 splicing isoforms were downregulated, while 426 were upregulated. Currently, we are focusing our attention on the pathways which are mostly affected by FUS depletion. In addition, to further characterize the FUS-depleted cell line we have performed growth proliferation and survival assays. From these experiments emerge that FUS-depleted cells display growth proliferation alteration. In order to explain this observation, we have tested different hypothesis (e.g. apoptosis, senescence or slow-down growth). We observed that FUS-depleted cells growth slower than controls. Currently, we are looking for putative candidate targets causing this phenotype. Finally, since MEFs and B-lymphocytes derived from FUS knockdown mice display major sensitivity to ionizing radiation and chromosomal aberrations [1,2], we are exploring the effects of DNA damage in FUS-depleted cells by monitoring important components of DNA Damage Response (DDR). Taken together, these studies may contribute to our knowledge of the role of FUS in these cellular processes and will allow us to draw a clearer picture of mechanisms of neurodegenerative diseases.

The FUS protein is required for cell proliferation

FUS/TLS (fused in sarcoma/translocated in liposarcoma) protein, a ubiquitously expressed and highly conserved RNA binding protein, has been linked to a variety of cellular processes from mRNA processing to DNA repair. However, the precise function of FUS is not well understood. Recently, mutations in the FUS gene have been identified in familial and sporadic patients of Amyotrophic Lateral Sclerosis, a fatal neurodegenerative disorder characterized by dysfunction and death of motor neurons. Based on the observation that some mutations in the FUS gene induce cytoplasmic accumulation of FUS aggregates, we decided to explore a loss-of-function situation (i.e. inhibition of FUS’ nuclear function) to unravel the role of this protein. To this purpose, we have generated a SH-SY5Y human neuroblastoma cell line which expresses a doxycycline induced shRNA targeting FUS that efficiently depletes the protein. In order to characterize this cell line, we have characterized the poly(A) fraction by RNA deep sequencing. Preliminary results show that FUS depletion affects both mRNA expression and alternative splicing. Upon FUS depletion 330 genes are downregulated and 81 are upregulated. We also found that 395 splicing isoforms were downregulated, while 426 were upregulated. Currently, we are focusing our attention on the pathways which are mostly affected by FUS depletion. In addition, we are currently characterizing how FUS depletion affects cell proliferation and survival. We find that the lack of FUS impairs cell proliferation but does not induce apoptosis. Finally, since MEFs and B-lymphocytes derived from FUS knockdown mice display major sensitivity to ionizing radiation and chromosomal aberrations [1,2], we are exploring the effects of DNA damage in FUS-depleted cells by monitoring important components of DNA Damage Response (DDR). Taken together, these studies may contribute to our knowledge of the role of FUS in these cellular processes and will allow us to draw a clearer picture of mechanisms of neurodegenerative diseases.

A rheological characterization of mashed potatoes enriched with soy protein isolate

The effect of the addition of soy protein isolate (SPI) (0, 15, 30, 45 and 60 g kg ) on viscoelastic properties, large deformation measurements and microstructure of fresh (FM) and frozen/thawed (F/TM) mashed potatoes was investigated. Rheological data showed weak gel behaviour for both FM and F/TM potatoes without and with added SPI together with a signi?cant decrease of system viscoelasticity (G and G ) with increasing SPI volume fraction, primarily attributed to the no interaction between the amylose/amylopectine matrix and the dispersed SPI particles or aggregates as revealed by scanning electron microscopy (SEM). Micrographs also showed that SPI formed white coarse aggregates. A freeze/thaw cycle produced a more signi?cant decrease in viscoelastic functions, due to superior aggregation of denatured SPI and reduced water activity. In F/TM samples, high correlations between small and large deformation measurements were found. Results may be useful for technological applications in SPI-enriched.

Colocalization of cell division proteins FtsZ and FtsA to cytoskeletal structures in living Escherichia coli cells by using green fluorescent protein

In the current model for bacterial cell division, FtsZ protein forms a ring that marks the division plane, creating a cytoskeletal framework for the subsequent action of other proteins such as FtsA. This putative protein complex ultimately generates the division septum. Herein we report that FtsZ and FtsA proteins tagged with green fluorescent protein (GFP) colocalize to division-site ring-like structures in living bacterial cells in a visible space between the segregated nucleoids. Cells with higher levels of FtsZ–GFP or with FtsA–GFP plus excess wild-type FtsZ were inhibited for cell division and often exhibited bright fluorescent spiral tubules that spanned the length of the filamentous cells. This suggests that FtsZ may switch from a septation-competent localized ring to an unlocalized spiral under some conditions and that FtsA can bind to FtsZ in both conformations. FtsZ–GFP also formed nonproductive but localized aggregates at a higher concentration that could represent FtsZ nucleation sites. The general domain structure of FtsZ–GFP resembles that of tubulin, since the C terminus of FtsZ is not required for polymerization but may regulate polymerization state. The N-terminal portion of Rhizobium FtsZ polymerized in Escherichia coli and appeared to copolymerize with E. coli FtsZ, suggesting a degree of interspecies functional conservation. Analysis of several deletions of FtsA–GFP suggests that multiple segments of FtsA are important for its localization to the FtsZ ring.

Stimulation of amyloid precursor protein synthesis by adrenergic receptors coupled to cAMP formation

Amyloid plaques in Alzheimer disease are primarily aggregates of Aβ peptides that are derived from the amyloid precursor protein (APP). Neurotransmitter agonists that activate phosphatidylinositol hydrolysis and protein kinase C stimulate APP processing and generate soluble, non-amyloidogenic APP (APPs). Elevations in cAMP oppose this stimulatory effect and lead to the accumulation of cell-associated APP holoprotein containing amyloidogenic Aβ peptides. We now report that cAMP signaling can also increase cellular levels of APP holoprotein by stimulating APP gene expression in astrocytes. Treatment of astrocytes with norepinephrine or isoproterenol for 24 h increased both APP mRNA and holoprotein levels, and these increases were blocked by the β-adrenergic antagonist propranolol. Treatment with 8-bromo-adenosine 3′,5′-cyclic monophosphate or forskolin for 24 h similarly increased APP holoprotein levels; astrocytes were also transformed into process-bearing cells expressing increased amounts of glial fibrillary acidic protein, suggesting that these cells resemble reactive astrocytes. The increases in APP mRNA and holoprotein in astrocytes caused by cAMP stimulation were inhibited by the immunosuppressant cyclosporin A. Our study suggests that APP overexpression by reactive astrocytes during neuronal injury may contribute to Alzheimer disease neuropathology, and that immunosuppressants can inhibit cAMP activation of APP gene transcription.

Transmembrane Insertion of the Toxoplasma gondii GRA5 Protein Occurs after Soluble Secretion into the Host Cell

The intracellular parasite Toxoplasma gondii resides within a specialized compartment, the parasitophorous vacuole (PV), that resists fusion with host cell endocytic and lysosomal compartments. The PV is extensively modified by secretion of parasite proteins, including the dense granule protein GRA5 that is specifically targeted to the delimiting membrane of the PV (PVM). We show here that GRA5 is present both in a soluble form and in hydrophobic aggregates. GRA5 is secreted as a soluble form into the PV after which it becomes stably associated with the PVM. Topological studies demonstrated that GRA5 was inserted into the PVM as a transmembrane protein with its N-terminal domain extending into the cytoplasm and its C terminus in the vacuole lumen. Deletion of 8 of the 18 hydrophobic amino acids of the single predicted transmembrane domain resulted in the failure of GRA5 to associate with the PVM; yet it remained correctly packaged in the dense granules and was secreted as a soluble protein into the PV. Collectively, these studies demonstrate that the secretory pathway in Toxoplasma is unusual in two regards; it allows soluble export of proteins containing typical transmembrane domains and provides a mechanism for their insertion into a host cell membrane after secretion from the parasite.

The Endogenous and Cell Cycle-dependent Phosphorylation of tau Protein in Living Cells: Implications for Alzheimer’s Disease

In Alzheimer’s disease the neuronal microtubule-associated protein tau becomes highly phosphorylated, loses its binding properties, and aggregates into paired helical filaments. There is increasing evidence that the events leading to this hyperphosphorylation are related to mitotic mechanisms. Hence, we have analyzed the physiological phosphorylation of endogenous tau protein in metabolically labeled human neuroblastoma cells and in Chinese hamster ovary cells stably transfected with tau. In nonsynchronized cultures the phosphorylation pattern was remarkably similar in both cell lines, suggesting a similar balance of kinases and phosphatases with respect to tau. Using phosphopeptide mapping and sequencing we identified 17 phosphorylation sites comprising 80–90% of the total phosphate incorporated. Most of these are in SP or TP motifs, except S214 and S262. Since phosphorylation of microtubule-associated proteins increases during mitosis, concomitant with increased microtubule dynamics, we analyzed cells mitotically arrested with nocodazole. This revealed that S214 is a prominent phosphorylation site in metaphase, but not in interphase. Phosphorylation of this residue strongly decreases the tau–microtubule interaction in vitro, suppresses microtubule assembly, and may be a key factor in the observed detachment of tau from microtubules during mitosis. Since S214 is also phosphorylated in Alzheimer’s disease tau, our results support the view that reactivation of the cell cycle machinery is involved in tau hyperphosphorylation.

A Novel Ras-interacting Protein Required for Chemotaxis and Cyclic Adenosine Monophosphate Signal Relay in Dictyostelium

We have identified a novel Ras-interacting protein from Dictyostelium, RIP3, whose function is required for both chemotaxis and the synthesis and relay of the cyclic AMP (cAMP) chemoattractant signal. rip3 null cells are unable to aggregate and lack receptor activation of adenylyl cyclase but are able, in response to cAMP, to induce aggregation-stage, postaggregative, and cell-type-specific gene expression in suspension culture. In addition, rip3 null cells are unable to properly polarize in a cAMP gradient and chemotaxis is highly impaired. We demonstrate that cAMP stimulation of guanylyl cyclase, which is required for chemotaxis, is reduced ∼60% in rip3 null cells. This reduced activation of guanylyl cyclase may account, in part, for the defect in chemotaxis. When cells are pulsed with cAMP for 5 h to mimic the endogenous cAMP oscillations that occur in wild-type strains, the cells will form aggregates, most of which, however, arrest at the mound stage. Unlike the response seen in wild-type strains, the rip3 null cell aggregates that form under these experimental conditions are very small, which is probably due to the rip3 null cell chemotaxis defect. Many of the phenotypes of the rip3 null cell, including the inability to activate adenylyl cyclase in response to cAMP and defects in chemotaxis, are very similar to those of strains carrying a disruption of the gene encoding the putative Ras exchange factor AleA. We demonstrate that aleA null cells also exhibit a defect in cAMP-mediated activation of guanylyl cyclase similar to that of rip3 null cells. A double-knockout mutant (rip3/aleA null cells) exhibits a further reduction in receptor activation of guanylyl cyclase, and these cells display almost no cell polarization or movement in cAMP gradients. As RIP3 preferentially interacts with an activated form of the Dictyostelium Ras protein RasG, which itself is important for cell movement, we propose that RIP3 and AleA are components of a Ras-regulated pathway involved in integrating chemotaxis and signal relay pathways that are essential for aggregation.

Decreased ability of HIV-1 Tat protein-treated accessory cells to organize cellular clusters is associated with partial activation of T cells

It has been shown in several animal models that HIV infection of accessory cells (ACs) plays an important role in development of AIDS. Here, we report that ACs treated with HIV-1 Tat protein (Tat-ACs) have a decreased ability to organize cellular aggregates as compared with untreated ACs, resulting in incomplete activation of T cells in responses to anti-CD3 mAb or staphylococcal enterotoxin B stimulation. The T cells failed to up-regulate adhesion molecules CD11a and CD2 on the cell surface and had reduced proliferative responses, as determined by [3H]thymidine incorporation, but they obtained lymphoblast-like morphology and expressed early activation antigens on the cell surface such as Fas and CD69 and interleukin 2 receptor, at comparable levels as those T cells undergoing a maximal proliferation. These results suggest that the Tat-AC-induced defect occurs in the late, but not in the early, phases of T cell activation. Normal expression of cell surface Fas antigen accompanied by defects in late activation thus may result in the susceptibility of these T cells to apoptosis. Our studies suggest that dysfunction, hyperactivation, and susceptibility to apoptosis, as observed with T cells isolated from HIV-infected individuals, may be, at least in part, a consequence of abnormal functions of ACs.