TATPred:a Bayesian method for the identification of twin arginine translocation pathway signal sequences

The twin arginine translocation (TAT) system ferries folded proteins across the bacterial membrane. Proteins are directed into this system by the TAT signal peptide present at the amino terminus of the precursor protein, which contains the twin arginine residues that give the system its name. There are currently only two computational methods for the prediction of TAT translocated proteins from sequence. Both methods have limitations that make the creation of a new algorithm for TAT-translocated protein prediction desirable. We have developed TATPred, a new sequence-model method, based on a Nave-Bayesian network, for the prediction of TAT signal peptides. In this approach, a comprehensive range of models was tested to identify the most reliable and robust predictor. The best model comprised 12 residues: three residues prior to the twin arginines and the seven residues that follow them. We found a prediction sensitivity of 0.979 and a specificity of 0.942.

Produção de nanopartículas contendo peptídeos bioativos de microalgas

A produção de peptídeos bioativos de distintas fontes de proteínas vem ganhando espaço na produção científica e tecnológica, despertando interesse do setor empresarial. Paralelamente a isso, devido à elevada concentração de proteínas na biomassa das microalgas Spirulina e Chlorella, estas apresentam grande potencial para a extração de biocompostos com alto valor agregado, como biopeptídeos de microalgas. As proteínas são uma importante fonte de peptídeos bioativos, mas estes não estão ativos na proteína precursora e devem ser liberados para que apresentem efeitos fisiológicos desejados. Essa liberação pode ser feita através de hidrólise enzimática a partir de proteases, sendo um dos métodos mais utilizados para a produção destes biocompostos. Dentro deste contexto, vários estudos vêm mostrando o uso da tecnologia por secagem em spray dryer para a obtenção de nanopartículas que contenham compostos bioativos, sendo, essa técnica, amplamente utilizada para transformar líquidos em pós, podendo ser aplicada em materiais sensíveis à temperatura. Este estudo teve como objetivo obter peptídeos bioativos através da reação enzimática, tendo como substrato a biomassa de Spirulina sp. LEB 18 e Chlorella pyrenoidosa e, na sequência, obter nanopartículas contendo os biopeptídeos. Primeiramente, foram testadas as 3 proteases comerciais (Protemax 580 L, Protemax N 200 e pepsina) para a produção de hidrolisados proteicos de microalgas, para isso foram realizados 3 delineamentos compostos centrais para cada microalga em estudo (Chlorella e Spirulina). Os delineamentos utilizados foram do tipo 23 com três repetições no ponto central, variando-se a concentração de enzima (5 a 10 U.mL-1), a concentração de substrato (5 a 10 %) e o tempo de reação (60 a 240 min). Após, realizou-se 2 delineamentos compostos rotacionais do tipo 22 com pontos centrais, um para cada microalga, utilizando-se para a hidrólise a enzima Protemax 580L (5 U.mL-1) variando-se a concentração de substrato e tempo de reação, para todos ensaios estudou-se a solubilidade, capacidade de retenção de água, atividade antioxidante e digestibilidade. Foi selecionado um ensaio para cada microalga, levando em conta os melhores resultados. Então nova hidrólise enzimática foi realizada sendo o sistema reacional composto pela enzima Protemax 580 L (5 U.mL-1) e pela biomassa de Spirulina sp. LEB 18 ou Chlorella pyrenoidosa (4% de proteína) durante tempo de 200 min. Os hidrolisados foram purificados por filtração a vácuo com membranas millipores de diferentes tamanhos (0,45; 0,2 e 0,1 µm) e por colunas com membrana vertical Amicon® Ultra 0.5 (3K e 10K), sendo que após cada etapa, foi realizado teste de atividade antioxidante pelos métodos de poder redutor, DPPH e ABTS, a fim de verificar a permanência da atividade antioxidante. Utilizou-se nano spray dryer Büchi modelo B 90 para a secagem das amostras, sendo o tamanho das partículas obtidas analisados por microscopia eletrônica de varredura (MEV). Por fim, conclui-se que a biomassa de microalgas pode ser utilizada como fonte de produção de peptídeos bioativos com elevada atividade antioxidante e que dentre as microalgas estudadas, Spirulina sp. LEB 18 apresentou melhores resultados, em todas as análises realizadas, quando comparada com Chlorella pyrenoidosa. Esse estudo, também visou utilizar a nanobiotecnologia para obtenção de nanoparículas contendo os biopeptídeos, para tal, utilizou-se o nano Buchi Spray Dryer B-90, o qual gerou partículas nanométricas de 14 a 18 nm para o hidrolisado de Spirulina e de 72 a 108 nm para o hidrolisado de Chlorella.

Evaluation of the transcriptomic response of an Alzheimer's disease murine model induced at different ages: searching for predictors

Alzheimer’s disease (AD) is a chronic, progressive neurodegenerative disease, characterized by the impairment of mnesic and cognitive functions, that represents the most frequent type of dementia in older people worldwide. Aging is the most important risk factor for the sporadic form of the pathology and it is associated to the progressive impairment of the proteostasis network. The endoplasmic reticulum (ER), the main cellular actor involved in proteostasis, appears significantly compromised in AD due to the accumulation of β-amyloid (Aβ) protein and phosphorylated-tau protein. Increasing proteins misfolding activates a specific cellular response known as Unfolded Protein response (UPR) which orchestrates the recovery of ER function. The aim of the present study was to investigate the role of UPR and aging process in a murine model of AD induced by intracerebroventricular (i.c.v.) injection of Aβ1-42 oligomers at 3 or 18 months. The oligomers injection in aged animals caused the increased of memory impairment, oxidative stress, and the depletion of glutathione reserve. Furthermore, the RNA-sequencing analysis was performed and the bioinformatic analysis showed the enrichment of several pathways involved in neurodegeneration and protein regulations. The following analysis highlighted the significant dysregulation of the three branches of the UPR, the protein kinase RNA-like ER kinase (PERK), inositol-requiring protein 1α (IRE1α) and activating transcription factor 6 (ATF-6). In turn, ER stress affected the PI3K/Akt/Gsk3β and MAPK/ERK pathways, highlighting Mapkapk5 as a potential marker of the neurodegenerative process, which regulation could lead to the definition of new pharmacological and neuroprotective strategies to counteract AD.

Protein aggregation containing beta-amyloid, alpha-synuclein and hyperphosphorylated tau in cultured cells of hippocampus, substantia nigra and locus coeruleus after rotenone exposure

Background: Protein aggregates containing alpha-synuclein, beta-amyloid and hyperphosphorylated tau are commonly found during neurodegenerative processes which is often accompanied by the impairment of mitochondrial complex I respiratory chain and dysfunction of cellular systems of protein degradation. In view of this, we aimed to develop an in vitro model to study protein aggregation associated to neurodegenerative diseases using cultured cells from hippocampus, locus coeruleus and substantia nigra of newborn Lewis rats exposed to 0.5, 1, 10 and 25 nM of rotenone, which is an agricultural pesticide, for 48 hours. Results: We demonstrated that the proportion of cells in culture is approximately the same as found in the brain nuclei they were extracted from. Rotenone at 0.5 nM was able to induce alpha-synuclein and beta amyloid aggregation, as well as increased hyperphosphorylation of tau, although high concentrations of this pesticide (over 1 nM) lead cells to death before protein aggregation. We also demonstrated that the 14kDa isoform of alpha-synuclein is not present in newborn Lewis rats. Conclusion: Rotenone exposure may lead to constitutive protein aggregation in vitro, which may be of relevance to study the mechanisms involved in idiopathic neurodegeneration.

Participation of kinin receptors on memory impairment after chronic infusion of human amyloid-beta 1-40 peptide in mice

Chronic infusion of human amyloid-beta 1-40 (A beta) in the lateral ventricle (LV) of rats is associated with memory impairment and increase of kinin receptors in cortical and hippocampal areas. Deletion of kinin B1 or B2 receptors abolished memory impairment caused by an acute single injection of A beta in the LV. As brain tissue and kinin receptors could unlikely react to acute or chronic administration of a similar quantity of A beta, we evaluated the participation of B1 or B2 receptors in memory impairment after chronic infusion of A beta. Male C57BI/6 J (wt), knock-out B1 (koB1) or B2 (koB2) mice (12 weeks of age) previously trained in a two-way shuttle-box and achieving conditioned avoidance responses (CAR, % of 50 trials) were infused with AB (550 pmol, 0.12 mu L/h, 28 days) or vehicle in the LV using a mini-osmotic pump. They were tested before the surgery (TO), 7 and 35 days after the infusion started (T7; T35). In T0, no difference was observed between CAR of the control (Cwt = 59.7 +/- 6.7%; CkoB1 = 46.7 +/- 4.0%; CkoB2 = 64.4 +/- 5.8%) and A beta (A beta wt = 66.0 +/- 3.0%; A beta koB1 = 66.8 +/- 8.2%; A beta koB2 = 58.7 +/- 5.9%) groups. In T7, A beta koB2 showed a significant decrease in CAR (41.0 +/- 8.6%) compared to the control-koB2 (72.8 +/- 2.2%, P <0.05). In T35, a significant decrease (P <0.05) was observed in A beta wt (40.7 +/- 3.3%) and A beta koB2 (41.2 +/- 10.7%) but not in the A beta koB1 (64.0 +/- 14.0%) compared to their control groups. No changes were observed in the controls at T35. We suggest that in chronic infusion of BA, B1 receptors could playan important role in the neurodegenerative process. Conversely, the premature memory impairment of koB2 suggests that it may be a protective factor. (C) 2009 Elsevier Ltd. All rights reserved.

Distinct expression pattern of microtubule-associated protein-2 in human oligodendrogliomas and glial precursor cells.

Microtubule-associated protein 2 (MAP2), a protein linked to the neuronal cytoskeleton in the mature central nervous system (CNS), has recently been identified in glial precursors indicating a potential role during glial development. In the present study, we systematically analyzed the expression of MAP2 in a series of 237 human neuroepithelial tumors including paraffin-embedded specimens and tumor tissue microarrays from oligodendrogliomas, mixed gliomas, astrocytomas, glioblastomas, ependymomas, as well as dysembryoplastic neuroepithelial tumors (DNT), and central neurocytomas. In addition, MAP2-immunoreactive precursor cells were studied in the developing human brain. Three monoclonal antibodies generated against MAP2A-B or MAP2A-D isoforms were used. Variable immunoreactivity for MAP2 could be observed in all gliomas with the exception of ependymomas. Oligodendrogliomas exhibited a consistently strong and distinct pattern of expression characterized by perinuclear cytoplasmic staining without significant process labeling. Tumor cells with immunoreactive bi- or multi-polar processes were mostly encountered in astroglial neoplasms, whereas the small cell component in neurocytomas and DNT was not labeled. These features render MAP2 immunoreactivity a helpful diagnostic tool for the distinction of oligodendrogliomas and other neuroepithelial neoplasms. RT-PCR, Western blot analysis, and in situ hybridization confirmed the expression of MAP2A-C (including the novel MAP2+ 13 transcript) in both oligodendrogliomas and astrocytomas. Double fluorescent laser scanning microscopy showed that GFAP and MAP2 labeled different tumor cell populations. In embryonic human brains, MAP2-immunoreactive glial precursor cells were identified within the subventricular or intermediate zones. These precursors exhibit morphology closely resembling the immunolabeled neoplastic cells observed in glial tumors. Our findings demonstrate MAP2 expression in astrocytic and oligodendroglial neoplasms. The distinct pattern of immunoreactivity in oligodendrogliomas may be useful as a diagnostic tool. Since MAP2 expression occurs transiently in migrating immature glial cells, our findings are in line with an assumed origin of diffuse gliomas from glial precursors.

Amyloid and non-amyloid forms of 5q31-linked corneal dystrophy resulting from kerato-epithelin mutations at Arg-124 are associated with abnormal turnover of the protein.

Mutations in kerato-epithelin are responsible for a group of hereditary cornea-specific deposition diseases, 5q31-linked corneal dystrophies. These conditions are characterized by progressive accumulation of protein deposits of different ultrastructure. Herein, we studied the corneas with mutations at kerato-epithelin residue Arg-124 resulting in amyloid (R124C), non-amyloid (R124L), and a mixed pattern of deposition (R124H). We found that aggregated kerato-epithelin comprised all types of pathological deposits. Each mutation was associated with characteristic changes of protein turnover in corneal tissue. Amyloidogenesis in R124C corneas was accompanied by the accumulation of N-terminal kerato-epithelin fragments, whereby species of 44 kDa were the major constituents of amyloid fibrils. R124H corneas with prevailing non-amyloid inclusions showed accumulation of a new 66-kDa species altogether with the full-size 68-kDa form. Finally, in R124L cornea with non amyloid deposits, we found only the accumulation of the 68-kDa form. Two-dimensional gels revealed mutation-specific changes in the processing of the full-size protein in all affected corneas. It appears that substitutions at the same residue (Arg-124) result in cornea-specific deposition of kerato-epithelin via distinct aggregation pathways each involving altered turnover of the protein in corneal tissue.

Fine structure of the vaccinia virus gene encoding the precursor of the major core protein 4 a.

A 6008 base pair fragment of the vaccinia virus DNA containing the gene for the precursor of the major core protein 4 a, which has been designated P4 a, was sequenced. A long open reading frame (ORF) encoding a protein of molecular weight 102,157 started close to the position where the P4 a mRNA had been mapped. Analysis of the mRNA by S1 nuclease mapping and primer extension indicated that the 5' end defined by the former method is not the true 5' end. This suggests that the P4 a coding region is preceded by leader sequences that are not derived from the immediate vicinity of the gene, similar to what has been reported for another late vaccinia virus mRNA. The sequenced DNA contained several further ORFs on the same, or opposite DNA strand, providing further evidence for the close spacing of protein-coding sequences in the viral genome.

Role of the cellular prion protein in oligodendrocyte precursor cell proliferation and differentiation in the developing and adult mouse CNS

There are numerous studies describing the signaling mechanisms that mediate oligodendrocyte precursor cell (OPC) proliferation and differentiation, although the contribution of the cellular prion protein (PrPc) to this process remains unclear. PrPc is a glycosyl-phosphatidylinositol (GPI)-anchored glycoprotein involved in diverse cellular processes during the development and maturation of the mammalian central nervous system (CNS). Here we describe how PrPc influences oligodendrocyte proliferation in the developing and adult CNS. OPCs that lack PrPc proliferate more vigorously at the expense of a delay in differentiation, which correlates with changes in the expression of oligodendrocyte lineage markers. In addition, numerous NG2-positive cells were observed in cortical regions of adult PrPc knockout mice, although no significant changes in myelination can be seen, probably due to the death of surplus cells.

Serum Amyloid A, C-reactive Protein and Retinal Microvascular Changes in Hypertensive Diabetic and Non-diabetic Individuals: An ASCOT Substudy

To study the association of the inflammatory markers serum amyloid A (SAA) and C-reactive protein (CRP) with retinal microvascular parameters in hypertensive individuals with and without type 2 diabetes.

Rpp2, an essential protein subunit of nuclear RNase P, is required for processing of precursor tRNAs and 35S precursor rRNA in Saccharomyces cerevisiae

RPP2, an essential gene that encodes a 15.8-kDa protein subunit of nuclear RNase P, has been identified in the genome of Saccharomyces cerevisiae. Rpp2 was detected by sequence similarity with a human protein, Rpp20, which copurifies with human RNase P. Epitope-tagged Rpp2 can be found in association with both RNase P and RNase mitochondrial RNA processing in immunoprecipitates from crude extracts of cells. Depletion of Rpp2 protein in vivo causes accumulation of precursor tRNAs with unprocessed introns and 5′ and 3′ termini, and leads to defects in the processing of the 35S precursor rRNA. Rpp2-depleted cells are defective in processing of the 5.8S rRNA. Rpp2 immunoprecipitates cleave both yeast precursor tRNAs and precursor rRNAs accurately at the expected sites and contain the Rpp1 protein orthologue of the human scleroderma autoimmune antigen, Rpp30. These results demonstrate that Rpp2 is a protein subunit of nuclear RNase P that is functionally conserved in eukaryotes from yeast to humans.

Temperature dependence of amyloid β-protein fibrillization

Fibrillogenesis of the amyloid β-protein (Aβ) is believed to play a central role in the pathogenesis of Alzheimer’s disease. Previous studies of the kinetics of Aβ fibrillogenesis showed that the rate of fibril elongation is proportional to the concentration of monomers. We report here the study of the temperature dependence of the Aβ fibril elongation rate constant, ke, in 0.1 M HCl. The rate of fibril elongation was measured at Aβ monomer concentrations ranging from 50 to 400 μM and at temperatures from 4°C to 40°C. Over this temperature range, ke increases by two orders of magnitude. The temperature dependence of ke follows the Arrhenius law, ke = A exp (−EA/kT). The preexponential factor A and the activation energy EA are ≈6 × 1018 liter/(mol·sec) and 23 kcal/mol, respectively. Such a high value of EA suggests that significant conformational changes are associated with the binding of Aβ monomers to fibril ends.

Chemical synthesis of the precursor molecule of the Aequorea green fluorescent protein, subsequent folding, and development of fluorescence

The present paper describes the total chemical synthesis of the precursor molecule of the Aequorea green fluorescent protein (GFP). The molecule is made up of 238 amino acid residues in a single polypeptide chain and is nonfluorescent. To carry out the synthesis, a procedure, first described in 1981 for the synthesis of complex peptides, was used. The procedure is based on performing segment condensation reactions in solution while providing maximum protection to the segment. The effectiveness of the procedure has been demonstrated by the synthesis of various biologically active peptides and small proteins, such as human angiogenin, a 123-residue protein analogue of ribonuclease A, human midkine, a 121-residue protein, and pleiotrophin, a 136-residue protein analogue of midkine. The GFP precursor molecule was synthesized from 26 fully protected segments in solution, and the final 238-residue peptide was treated with anhydrous hydrogen fluoride to obtain the precursor molecule of GFP containing two Cys(acetamidomethyl) residues. After removal of the acetamidomethyl groups, the product was dissolved in 0.1 M Tris⋅HCl buffer (pH 8.0) in the presence of DTT. After several hours at room temperature, the solution began to emit a green fluorescence (λmax = 509 nm) under near-UV light. Both fluorescence excitation and fluorescence emission spectra were measured and were found to have the same shape and maxima as those reported for native GFP. The present results demonstrate the utility of the segment condensation procedure in synthesizing large protein molecules such as GFP. The result also provides evidence that the formation of the chromophore in GFP is not dependent on any external cofactor.

Protein C-Mannosylation Is Enzyme-catalysed and Uses Dolichyl-Phosphate-Mannose as a Precursor

C-mannosylation of Trp-7 in human ribonuclease 2 (RNase 2) is a novel kind of protein glycosylation that differs fundamentally from N- and O-glycosylation in the protein-sugar linkage. Previously, we established that the specificity determinant of the acceptor substrate (RNase 2) consists of the sequence W-x-x-W, where the first Trp becomes C-mannosylated. Here we investigated the reaction with respect to the mannosyl donor and the involvement of a glycosyltransferase. C-mannosylation of Trp-7 was reduced 10-fold in CHO (Chinese hamster ovary) Lec15 cells, which are deficient in dolichyl-phosphate-mannose (Dol-P-Man) synthase activity, compared with wild-type cells. This was not a result of a decrease in C-mannosyltransferase activity. Rat liver microsomes were used to C-mannosylate the N-terminal dodecapeptide from RNase 2 in vitro, with Dol-P-Man as the donor. This microsomal transferase activity was destroyed by heat and protease treatment, and displayed the same acceptor substrate specificity as the in vivo reaction studied previously. The C-C linkage between the indole and the mannosyl moiety was demonstrated by tandem electrospray mass spectrometry analysis of the product. GDP-Man, in the presence of Dol-P, functioned as a precursor in vitro with membranes from wild-type but not CHO Lec15 cells. In contrast, with Dol-P-Man both membrane preparations were equally active. It is concluded that a microsomal transferase catalyses C-mannosylation of Trp-7, and that the minimal biosynthetic pathway can be defined as: Man –> –> GDP-Man –> Dol-P-Man –> (C2-Man-)Trp.

Inhibiting transthyretin amyloid fibril formation via protein stabilization

Transthyretin (TTR) amyloid fibril formation is observed systemically in familial amyloid polyneuropathy and senile systemic amyloidosis and appears to be the causative agent in these diseases. Herein, we demonstrate conclusively that thyroxine (10.8 μM) inhibits TTR fibril formation efficiently in vitro and does so by stabilizing the tetramer against dissociation and the subsequent conformational changes required for amyloid fibril formation. In addition, the nonnative ligand 2,4,6-triiodophenol, which binds to TTR with slightly increased affinity also inhibits TTR fibril formation by this mechanism. Sedimentation velocity experiments were employed to show that TTR undergoes dissociation (linked to a conformational change) to form the monomeric amyloidogenic intermediate, which self-assembles into amyloid in the absence, but not in the presence of thyroxine. These results demonstrate the feasibility of using small molecules to stabilize the native fold of a potentially amyloidogenic human protein, thus preventing the conformational changes, which appear to be the common link in several human amyloid diseases. This strategy and the compounds resulting from further development should prove useful for critically evaluating the amyloid hypothesis—i.e., the putative cause-and-effect relationship between TTR amyloid deposition and the onset of familial amyloid polyneuropathy and senile systemic amyloidosis.