New mitochondrial and nuclear evidences support recent demographic expansion and an atypical phylogeographic pattern in the Spittlebug Philaenus spumarius (Hemiptera, Aphrophoridae)

Copyright: © 2014 Rodrigues et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Cholesterol 24S-Hydroxylase overexpression inhibits the liver X receptor (LXR) pathway by activating small guanosine triphosphate-binding proteins (sGTPases) in neuronal cells

The neuronal-specific cholesterol 24S-hydroxylase (CYP46A1) is important for brain cholesterol elimination. Cyp46a1 null mice exhibit severe deficiencies in learning and hippocampal long-term potentiation, suggested to be caused by a decrease in isoprenoid intermediates of the mevalonate pathway. Conversely, transgenic mice overexpressing CYP46A1 show an improved cognitive function. These results raised the question of whether CYP46A1 expression can modulate the activity of proteins that are crucial for neuronal function, namely of isoprenylated small guanosine triphosphate-binding proteins (sGTPases). Our results show that CYP46A1 overexpression in SH-SY5Y neuroblastoma cells and in primary cultures of rat cortical neurons leads to an increase in 3-hydroxy-3-methyl-glutaryl-CoA reductase activity and to an overall increase in membrane levels of RhoA, Rac1, Cdc42 and Rab8. This increase is accompanied by a specific increase in RhoA activation. Interestingly, treatment with lovastatin or a geranylgeranyltransferase-I inhibitor abolished the CYP46A1 effect. The CYP46A1-mediated increase in sGTPases membrane abundance was confirmed in vivo, in membrane fractions obtained from transgenic mice overexpressing this enzyme. Moreover, CYP46A1 overexpression leads to a decrease in the liver X receptor (LXR) transcriptional activity and in the mRNA levels of ATP-binding cassette transporter 1, sub-family A, member 1 and apolipoprotein E. This effect was abolished by inhibition of prenylation or by co-transfection of a RhoA dominant-negative mutant. Our results suggest a novel regulatory axis in neurons; under conditions of membrane cholesterol reduction by increased CYP46A1 expression, neurons increase isoprenoid synthesis and sGTPase prenylation. This leads to a reduction in LXR activity, and consequently to a decrease in the expression of LXR target genes.

Electrochemical studies on small electron transfer proteins using membrane electrodes

Journal of Electroanalytical Chemistry 541 (2003) 153-162

Structural and mechanistic studies of iron containing proteins

Dissertação apresentada para obtenção do Grau de Doutor em Bioquímica, ramo de Bioquímica-Física, pela Universidade Nova de Lisboa, Faculdade de Ciências e Tecnologia

Molybdenum-containing proteins from Sulphate Reducing Bacteria: studying proteins with novel cofactors and revealing new features of old enzymes

Dissertação apresentada para a obtenção do Grau de Doutor em Bioquímica, especialidade de Bioquímica-Física pela Universidade Nova de Lisboa, Faculdade de Ciências e Tecnologia

Elution of proteins from polyacrylamide eels: a simple and economic procedure

A simplified methodology for the quantitative electroelution of proteins from polyacrylamide gels is described. After staining with Coomassie Brilliant Blue R 250, the identified bands are excised from the gel and the proteins eluted using a procedure developed for use in conventional tube gel electrophoresis equipment.

A regulated response to mitochondrial dysfunction modulates longevity and rates of living

Aging is a long-standing biological question of tremendous social and cultural importance. Despite this, only in the last 15 years has biology started to make significant progress in understanding the underlying mechanisms that regulate aging. This progress stemmed mainly from the use of model organisms, which allowed the discovery of several genes directly modulating longevity. Interestingly, several of these longevity genes are necessary for normal mitochondrial function, and disruption of their activity delays the aging process. This is somewhat paradoxical, considering the importance of cellular respiration for energy production and viability of eukaryotic organisms. One possible rationalization for this is that by decreasing cellular respiration, reactive oxygen species (ROS) generation is also reduced, and in that way, cellular decay and aging are delayed.(...)

Non-enzymatic assay for glucose by using immobilized whole-cells of E. coli containing glucose binding protein fused to fluorescent proteins

Glucose monitoring in vivo is a crucial issue for gaining new understanding of diabetes. Glucose binding protein (GBP) fused to two fluorescent indicator proteins (FLIP) was used in the present study such as FLIP-glu- 3.2 mM. Recombinant Escherichia coli whole-cells containing genetically encoded nanosensors as well as cell-free extracts were immobilized either on inner epidermis of onion bulb scale or on 96-well microtiter plates in the presence of glutaraldehyde. Glucose monitoring was carried out by Förster Resonance Energy Transfer (FRET) analysis due the cyano and yellow fluorescent proteins (ECFP and EYFP) immobilized in both these supports. The recovery of these immobilized FLIP nanosensors compared with the free whole-cells and cell-free extract was in the range of 50–90%. Moreover, the data revealed that these FLIP nanosensors can be immobilized in such solid supports with retention of their biological activity. Glucose assay was devised by FRET analysis by using these nanosensors in real samples which detected glucose in the linear range of 0–24 mM with a limit of detection of 0.11 mM glucose. On the other hand, storage and operational stability studies revealed that they are very stable and can be re-used several times (i.e. at least 20 times) without any significant loss of FRET signal. To author's knowledge, this is the first report on the use of such immobilization supports for whole-cells and cell-free extract containing FLIP nanosensor for glucose assay. On the other hand, this is a novel and cheap high throughput method for glucose assay.

Induction of iron regulated proteins during normal growth of Neisseria meningitidis in a chemically defined medium

The expression of iron regulated proteins (IRPs) in vitro has been obtained in the past by adding iron chelators to the culture after bacterial growth, in the presence of an organic iron source. We have investigated aspects concerning full expression of the meningococcal IRPs during normal growth, in defined conditions using Catlin medium, Mueller Hinton and Tryptic Soy Broth (TSB). The expression of IRPs varied between different strains with respect to Ethylenediamine Di-ortho-Hidroxy-phenyl-acetic acid (EDDA) concentrations, and according to culture medium, and also between different lots of TSB. For each strain, a specific set of IRPs were expressed and higher EDDA concentrations, or addition of glucose, or use of different culture media did not resulted in a differential expression of IRPs. We were not able to grow N. meningitidis under normal growth conditions using Desferal. We looked for a good yield of outer membrane vesicles (OMVs) expressing IRPs in iron-deficient Catlin medium containing EDDA and Hemin. Culture for 32 h at 30ºC after growing for 16 h at 37ºC supported good bacterial growth. Bacterial lysis was noted after additional 24 h at 30ºC. Approximately 4 times more OMVs was recoverable from a culture supernatant after 24 h at 30ºC than from the cells after 16 h at 37ºC. The IRP were as well expressed in OMVs from culture supernatant obtained after 24 h at 30ºC as from the cells after 16 h at 37ºC.

Defective protein folding and function in metabolic disorders: studies on the mitochondrial flavoenzyme ETF

Dissertation presented to obtain the PhD degree in Biochemistry at the Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa

A proteomic approach toward the selection of proteins with enhanced intrinsic conformational stability

Journal of Proteome Research (2006)5: 2720-2726

Role of a novel disulfide bridge within the all-beta fold of soluble Rieske proteins

Journal of Biological Inorganic Chemistry (2010)15: 271-281

Natural and amyloid self-assembly of S100 proteins: structural basis of functional diversity

The S100 proteins are 10-12 kDa EF-hand proteins that act as central regulators in a multitude of cellular processes including cell survival, proliferation, differentiation and motility. Consequently, many S100 proteins are implicated and display marked changes in their expression levels in many types of cancer, neurodegenerative disorders, inflammatory and autoimmune diseases. The structure and function of S100 proteins are modulated by metal ions via Ca2+ binding through EF-hand motifs and binding of Zn2+ and Cu2+ at additional sites, usually at the homodimer interfaces. Ca2+ binding modulates S100 conformational opening and thus promotes and affects the interaction with p53, the receptor for advanced glycation endproducts and Toll-like receptor 4, among many others. Structural plasticity also occurs at the quaternary level, where several S100 proteins self-assemble into multiple oligomeric states, many being functionally relevant. Recently, we have found that the S100A8/A9 proteins are involved in amyloidogenic processes in corpora amylacea of prostate cancer patients, and undergo metal-mediated amyloid oligomerization and fibrillation in vitro. Here we review the unique chemical and structural properties of S100 proteins that underlie the conformational changes resulting in their oligomerization upon metal ion binding and ultimately in functional control. The possibility that S100 proteins have intrinsic amyloid-forming capacity is also addressed, as well as the hypothesis that amyloid self-assemblies may, under particular physiological conditions, affect the S100 functions within the cellular milieu.